Preterm labour, resulting in delivery before 37 weeks, occurs in 5-10% of all pregnancies and is the major cause of perinatal morbidity and mortality in the developed world. Various tocolytic agents that modulate uterine smooth muscle contractility, β-adrenoceptor agonists, nifedipine, magnesium sulphate and prostaglandin synthase inhibitors have been trialed and either delay labour by <72 h and/or have serious cardiovascular effects on both the mother and fetus. We have investigated the mechanisms underlying the reduced myometrial contractility evoked by β-adrenoceptor activation in pregnant sheep with a view to identifying potential new targets for prolonging pregnancy in the event of preterm labour. Tissues were collected from pregnant sheep under anaesthesia initially established with sodium thiopentone (20 mg kg^-1 I.V.) and maintained with isoflurane (2% in oxygen) applied by intermittent positive pressure ventilation. Intracellular microelectrode recordings from strips of sheep myometrium (late pregnancy, not in labour) revealed that spontaneous contractions were associated with bursts of action potentials. Salbutamol (0.01-1 μM) evoked concentration-dependent membrane hyperpolarization (to −80 mV at 0.5 μM) which prevented action potential discharge. Salbutamol-evoked hyperpolarizations were reduced to 7±3% (n=11) of control by glibenclamide (1 μM, blocker of K_ATP channels), and fully blocked by glibenclamide plus ouabain (10 μM, blocker of Na⁺/K⁺ ATPase). Activation of K_ATP channels using PCO400, evoked concentration-dependent hyperpolarization (to −85 mV). The amplitude of this hyperpolarization was not influenced by iberiotoxin (50-100 nM) which blocks large conductance, Ca²⁺-activated K⁺ (BKCa) channels. Salbutamol-induced hyperpolarization was reduced (to 64±5% of control, n=7) by inhibitors of protein kinase A, Rp-8-Cl cAMPS, Rp-8-CPT cAMPS and H89. Membrane hyperpolarization was also elicited by forskolin (0.1 μM, adenylyl cyclase activator), Sp-cBIMPS (50 μM, protein kinase A activator), and by IBMX (50 μM, phosphodiesterase inhibitor). Recordings from patches isolated from myometrial cells revealed the presence of K⁺-selective ion channels with a conductance of 240 pS (in a symmetrical 130 mM K⁺ gradient) which were readily activated by membrane potential, internal Ca²⁺ (23 nM to 1.4 μM) or NS1619 (30 μM) and blocked by 50-100 nM iberiotoxin, characterizing them as BKCa channels. Salbutamol (0.1 μM 10-20 min) applied to the bath or in the pipette increased BKCa channel activity in cell-attached patches due to a leftward shift in their voltage range of activation. In the presence of iberiotoxin, salbutamol and PCO400 activated an additional K⁺ selective ion channel with a single channel conductance of 40 pS which was blocked by glibenclamide (1 μM). These results demonstrate that stimulation of β-adrenoceptors in sheep myometrium leads to the activation, not only of BKCa channels, as previously reported by several laboratories, but also, to an hitherto undescribed activation of KATP channels. These K_ATP channels contribute to the resting membrane potential in myometrium and activation of K_ATP channels appears to provide the major membrane conductance increase underlying the hyperpolarization induced by β-adrenoceptor activation. In contrast BKCa channel activation appeared responsible for the reduced action potential amplitudes recorded in the presence of salbutamol. While the adenylyl cyclase/protein kinase A second messenger pathway may contribute to the hyperpolarization in response to β-adrenoceptor activation, it is unlikely to be the only mechanism involved.