We report the effect of acute estrogen treatment in the expression of muscarinic acetylcholine receptors (mAChRs) in myometrium. Strips were obtained from rats in estrus (control) and treated with estrogen, 24 h before the experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed and m2, m3 and m5 mAChR mRNA subtypes were detected in myometrium from both groups. [ 3H]Quinuclidinyl benzilate ([ 3HQNB]) binding studies indicated that estrogen treatment did not change the affinity and density of mAChRs in myometrial membranes. Displacement curves of [ 3HQNB] with different mAChRs antagonists indicated a one-site fit for all antagonists tested. Comparison of p K i values indicated a significant correlation to M 2-mAChR subtype. Functional studies, however, showed that estrogen treatment increased myometrium sensitivity to carbachol and the calculated apparent affinity values were significantly correlated to M 3-mAChR. Furthermore, the pharmacological profile of the two populations of mAChR was not affected by estrogen. In conclusion, these results provide evidence for the presence of M 2- and M 3-mAChR, at the mRNA and protein level, in the rat myometrium and indicate that estrogen induces an increase in myometrial responsiveness to mAChR agonists.