The aim of the present study was to identify the muscarinic acetylcholine receptor (mAChR) mRNA subtypes in the rat seminal vesicle. Furthermore, the mAChR subtypes involved in the contraction of the seminal vesicle were also explored. Reverse transcriptase-polymerase chain reaction (PCR) was performed and five PCR products corresponding to M1-M5 mAChR mRNA subtypes were detected in this tissue. Functional pharmacological studies indicated that the rank order of mAChR antagonists in blocking the contractile effects of carbachol was p-fluoro-hexahydro-sila-difenidol (pF-HHSiD) >> tropicamide > methoctramine = pirenzepine. This antagonist profile indicates that M3 mAChR subtype is predominantly involved in the seminal vesicle contraction. Furthermore, immunohistochemical studies confirmed the presence of the M3 mAChR subtype in the smooth muscle layers. M2 mAChR subtype was also immunolocalized in smooth muscle cells and may be involved in the contraction of this tissue. The presence of M2 and M3 mAChR subtypes in the epithelial cells suggests that these receptors could be involved in the protein secretion. Taken together, the cholinergic neurotransmitter may be a factor controlling contractility and protein secretion in this tissue.