1 In many smooth muscle tissues a minor M 3 -muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M 2 -mACh receptor population. However, this is not the case for guinea-pig uterus where radioligand binding and functional studies exclude a dominant role for M 3 -mACh receptors. 2 Using tissue from animals pre-treated with diethylstilboestrol, estimates of antagonist a nity were made before and after selective alkylation procedures, together with estimates of agonist a nity to characterise the mACh receptor population mediating carbachol-induced contraction of guinea-pig isolated uterus. 3 Antagonist a nity estimates made at`protected' receptors were not signi®cantly di erent from those made in untreated tissues. However all estimations were signi®cantly di erent from those reported in guinea-pig ileum and atria. The rank order of a nities were atropine4zamifenacin=tripitramine4 methoctramine. Carbachol-induced contractions were insensitive to the M 4 -selective muscarinic toxin MTx-3, or PD102807 (0.1 mM) ruling out a role for M 4 -mACh receptors. 4 The agonist a nity value for L-660,863, a putative`M 2 -selective' agonist of 5.44+0.30 (n=6) was signi®cantly di erent from that reported in guinea-pig atria. In contrast, the pK A value for carbachol (4.22+0.17; n=8) agrees with that reported for guinea-pig ileum. 5 Carbachol-induced contractions were insensitive to pertussis toxin although carbachol-induced inhibition of forskolin-stimulated cyclic AMP production was attenuated, ruling out the involvement of G i -proteins in contraction. 6 Radioligand binding studies revealed a K D for N-[ 3 H]-methylscopolamine of 0.12+0.05 nM and a B max of 147+18 fmol mg protein 71 . Antagonist a nity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M 2 -mACh receptors. 7 These data suggest a small population of mACh receptors with an atypical operational pro®le which can not be distinguished using radioligand binding studies may mediate carbachol-induced contraction of guinea-pig isolated uterus.