Aim: The identity of muscarinic acetylcholine receptors (mAchR) involved in cholinergic-mediated contraction of the guinea pig gallbladder has been a matter of debate. Different groups have suggested the involvement of M-1, M-2, M-3, or M-4 receptor subtypes in the contraction of this tissue. The objective of this study was to identify the mAchR subtypes expressed in the guinea pig gallbladder by RT-PCR. Methods: Total RNA prepared from frozen guinea pig gallbladder tissue was amplified by using specific primers for the M-1-M-4 receptor subtypes. Results: M-2, M-3, and M-4 transcripts were detected in the following rank order: M-4 > M-2 > M-3. We were unable to demonstrate the expression of the M, receptor subtype in this tissue. Conclusions: Our results are in agreement with our previous binding and functional data.