The aim of this study was to investigate the possible interaction of L-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). L-cysteine (endogenous H2S substrate; 10(-6)-10(-3) M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10(-6)-10(-3) M) and acetylcholine (10(-9)-10(-4) M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H2S were reduced by non-selective mAChR antagonist atropine (5 x 10(-5) M), selective M-1 mAChR antagonist pirenzepine (5 x 10(-5) M) and selective M-3 mAChR antagonist 4-DAMP (10(-7) M) but not by selective M-2 mAChR antagonist AF-DX 116 (10(-6) M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor D,L-propargylglycine (PAG, 10(-2) M) and cystathionine-beta-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10(-3) M). L-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H2S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by L-cysteine but not by exogenous H2S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M-1 and M-3 mAChRs contributes to relaxant effect mediated by endogenous H2S but at same time L-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of L-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded. (C) 2017 Elsevier Inc. All rights reserved.