The aim of the present study was to characterize the muscarinic acetylcholine receptor subtypes present in the caput and cauda of rat epididymis. The specific binding of [³H]quinuclidinyl benzilate ([³H]QNB) to epididymal membranes was time dependent, temperature dependent, and saturable. The cauda epididymis showed higher affinity to [³H]QNB and higher muscarinic receptor density when compared to the caput region. The [³H]QNB binding was tested in competition studies with different muscarinic receptor antagonists. Each antagonist tested displaced [³H]QNB bound to caput and cauda epididymal membrane with similar affinity. Correlation among the negative logarithm of inhibition constant values (pK_i) for these antagonists obtained in the epididymis with their correspondent published pK_i values obtained in tissues that expressed each receptor subtype (M₁, M₂, M₃, and M₄) indicated that the muscarinic receptors present in caput and cauda epididymis belong to the muscarinic M₂ receptor subtype. When reverse transcription-polymerase chain reaction was used to identify muscarinic receptor mRNA subtypes in the epididymis, only m2 transcripts were detected in the caput region, while both m2 and m3 mRNA subtypes were observed in the cauda region. In conclusion, these results demonstrate that muscarinic receptors are present in the rat epididymis, with expression levels dependent on the region of the epididymis analyzed. Thus, the cholinergic neurotransmitter in the epididymis may be a factor controlling contractility and/or the luminal fluid microenvironment.