The mechanisms underlying pacemaker activity in the sino-atrial (SA) node remain controversial, with some giving greatest prominence to If (1), others emphasising a ‘calcium clock’ (2), and some favouring membrane currents other than If (3). The aim of the present experiments was to explore the possible dependence of If on cytosolic Ca2+, including that released from the sarcoplasmic reticulum (SR). All experiments were carried out on mouse spontaneously beating atrial preparations with intact SA node using methods similar to those described in (4). All observations are presented as mean ± SEM, and P<0.05 for all reported changes (with Students t test or Analysis of Variance as appropriate). ZD7288 applied at 1 μM to block If caused a reduction of spontaneous rate from 505 ± 24 to 228 ± 24 bpm (a reduction of 55 ± 4 % expressed as % resting rate). Exposure to ryanodine (3 μM to interfere with SR Ca2+ release) reduced beating rate, and under these conditions the effects of ZD7288 on spontaneous rate were reduced: ZD7288 reduced rate from 197 ± 40 bpm (ryanodine alone) to 148 ± 40 bpm (ryanodine and ZD7288), a reduction of 27 ± 12%. SR function is also suppressed by cyclopiazonic acid (CPA) to inhibit Ca2+ re-uptake by the SR. CPA (100 μM) reduced spontaneous beating rate, and the effects of ZD7288 were reduced in the presence of CPA: ZD7288 reduced spontaneous rate from 213 ± 13 bpm (CPA alone) to 147 ± 9 bpm (CPA and ZD7288), a reduction of 19 ± 4%. In the presence of ZD7288, the log(concentration)-response curve for effects of isoprenaline on spontaneous rate showed a reduced slope and maximum response. Addition of CPA in the presence of ZD7288, or of ryanodine in the presence of ZD7288 both further reduced the slope and maximum response of the log(concentration)-response curve for isoprenaline. These observations are consistent with a role for Ca2+ released from the SR in regulating If and therefore the rate of spontaneous beating of mouse SA node. Ca2+ released from the SR may stimulate adenylyl cyclases AC1 and AC8 that are present in SA node (5). The reduced slope and maximum of the log(concentration)-response curve for isoprenaline after ZD7288 is consistent with modulation of If during beta-adrenoceptor stimulation. However, since interference with SR function (with either ryanodine or CPA) caused a further depression of the slope and maximum of the log(concentration)-response curve for isoprenaline even when If was substantially blocked, there appears to be an additional contribution of SR-derived Ca2+ to the effects of β-adrenoceptor stimulation on spontaneous rate that is independent of If.