Over slightly more than a decade, particular attention has been focussed on whether spontaneous pacemaker activity in the sino-atrial (SA) node arises primarily from the voltage-dependent properties of ion channels in the cell membrane or whether cytosolic Ca²⁺ plays a significant role in regulating this activity. The purpose of this presentation is to briefly review progress over the last decade and to present recent observations from our laboratory that indicate substantial complexity in the many roles for cytosolic Ca²⁺ in regulating pacemaker activity. Initial work focussed on Ca²⁺ release from the sarcoplasmic reticulum, but it is clear that even in the absence of a functional SR cytosolic Ca²⁺ may still regulate pacemaker activity. Our observations continue to support a role for Ca²⁺ release from the SR in the absence of neurotransmitters and hormones, and the importance of this SR-released Ca²⁺ is heightened, for example, following β-adrenoceptor stimulation. Under the conditions of our experiments, application of ryanodine to block Ca²⁺ release and/or cyclopiazonic acid or thapsigargin to block Ca²⁺ uptake into the SR slows but does not stop spontaneous activity. Our observations support a continuing importance of cytosolic Ca²⁺ in regulating pacemaker activity when SR function is suppressed (see below). At least part of the role of the SR-released Ca²⁺ is to provoke electrogenic Na⁺/Ca²⁺ exchange (NCX) currents at the foot of the action potential, perhaps associated with Ca²⁺ ‘sparks’. However, under the conditions of our experiments, we detect sparks as separable events in only about 30% of guinea-pig SA-node cells, and in any case there may be additional roles of the subsarcolemmal Ca²⁺. Cytosolic Ca²⁺ (derived from the SR or from entry through the surface membrane) may play a role by directly influencing membrane transporters (e.g. NCX), via Ca²⁺-calmodulin-dependent protein kinase (CaMKII) or via other actions mediated by calmodulin. Our recent observations support the fundamental importance of NCX in maintaining pacemaker activity, since suppression of this pathway by rapid switch to low Na⁺ solutions (with Li⁺ as replacement) caused immediate cessation of beating before there was time for the secondary effects that are expected to occur following changes in cytosolic Ca²⁺ when this essential pathway for Ca²⁺ balance is inhibited. Depolarizing ionic currents via NCX could arise from the conventional electrogenic 3Na⁺:1Ca²⁺ mode of NCX or from a recently proposed Na⁺ ‘leak’ pathway through the same protein. Another potentially important player in pacemaker activity is the I_f current that is activated by hyperpolarization and regulated by cytosolic cAMP. Our recent observations shed light on regulation of this pathway by cytosolic Ca²⁺ and provide a possible resolution of the controversy about the role of this pathway in the absence of β-adrenoceptor stimulation. Evidence for the presence of Ca²⁺-stimulated adenylyl cyclase, AC1, in SA node but not ventricular muscle was found both using PCR to detect mRNA and using Western blotting to detect a protein of the expected molecular mass. Immunocytochemistry showed localisation of AC1 at the surface membrane. Functional electrophysiological studies supported the hypothesis that cytosolic Ca²⁺ activates AC1 to catalyse the formation of cAMP which in turn shifts activation of I_f in the depolarizing direction so that there is more I_f current at the pacemaker range of potentials. A further point is that conventional voltage-clamp protocols may lead to lower cytosolic Ca²⁺ than would be the case in a beating SA node cell and therefore to underestimation of the I_f current. In addition, the significance of the presence of AC1 at the surface membrane may be much wider than its importance for I_f. Stimulation of AC1 by subsarcolemmal Ca²⁺ may also lead to activation of PKA and phosphorylation of other channels including Ca²⁺ and K⁺ channels. The effects of cytosolic Ca²⁺ on enzymes are not expected to be confined to AC1 since there will be activation of CaMKII and there may also be additional effects on yet more Ca²⁺-regulated enzymes. For example, NO shows complex biphasic effects on pacemaker activity and some enzymes in the proposed pathways are Ca²⁺ sensitive. There is considerable potential for interaction and feedback in all these pathways which will also depend on the balance between phosphorylating enzymes and phosphatases (that can also be Ca²⁺ sensitive). The above arguments show the complex roles so far identified for cytosolic Ca²⁺ in the pacemaker activity of SA node. However, even greater complexity may be revealed if endogenous substances such as IP₃ in turn modulate this Ca²⁺.