The cholinergic system participates in the regulation of masculine sexual behavior, mainly through the muscarinic system. Recently, muscarinic receptors have been subdivided into at least two subtypes, M1 and M2, according to their differential affinity for pirenzepine. In this study, we analyzed the possible participation of the M1 muscarinic receptor subtype on masculine sexual behavior regulation. In the first experiment, trihexyphenidyl, a specific M1 antagonist, was administered to experienced adult male rats in a wide range of doses (from 0.1 to 6.4 mg/kg). No modification was observed in any of the male sexual behavior parameters recorded, with the exception of the highest dose at which an increase of the intromission frequency and a decrease of the ejaculation frequency were observed. In the second experiment, trihexyphenidyl was administered in several doses (from 0.2 to 1.6 mg/kg), before the administration of oxotremorine, a muscarinic agonist, at a dose that readily facilitates masculine sexual behavior. Trihexyphenidyl completely prevented the facilitatory effects of oxotremorine even at the smallest dose used. These results strongly suggest that the Ml muscarinic receptor subtype participates in the cholinergic facilitation of masculine sexual behavior.