The effects of acetylcholine on isolated uterine artery rings of non-pregnant and pregnant guinea-pigs were investigated. Acetylcholine induced a concentration- and endothelium-dependent relaxation of both types of vessels, with similar pD2 (non-pregnant: pD2 = 7.66; pregnant: pD2 = 7.59) and maximal response values. The pKA values (non-pregnant vs. pregnant) of acetylcholine were 6.17 vs. 6.09. The occupancy response relationship was non-linear since the half-maximal response to acetylcholine (non-pregnant vs. pregnant) was obtained with 2.86 vs. 2.91% receptor occupancy. In quiescent preparations, the muscarinic receptor antagonists, atropine, pirenzepine, N,N'-bis[6-[(2-methoxybenzyl)amino]hexyl]-1,8-octane-diamine tetrahydrochloride (methoctramine) and para-fluoro-hexahydro-sila-difenidol (pFHHSiD), produced parallel rightward shifts of the curves for acetylcholine and the slopes of the Schild plots were not significantly different from unity. The plots constrained to a slope of unity gave the following -log KB values (non-pregnant vs. pregnant): atropine (9.68 vs. 9.75), pirenzepine (6.75 vs. 6.53), methoctramine (6.13 vs. 6.23) and pFHHSiD (7.88 vs. 7.96). It is concluded that, in guinea-pig uterine arteries, acetylcholine induces endothelium-dependent relaxation and acts as a full agonist, regardless of pregnancy status. We suggest that the relaxation induced by acetylcholine, in either non-pregnant or pregnant guinea-pig uterine artery, is mediated via the M3 subtype of muscarinic receptors.