In the present study, we measured acetylcholine (ACh) released from rabbit detrusor smooth muscle strips induced by electrical field stimulation (EFS) using high-performance liquid chromatography coupled with microdialysis procedure. There were frequency- and duration-dependent increases in contractile response and ACh release. There was a significant, but not simple correlation between EFS-induced contraction and ACh release. Atropine caused a decrease and increase in the contractile response and ACh release, respectively. Pretreatment with propranolol increased ACh release, but pretreatment with phentolamine had no significant effect. These results demonstrate that this method is applicable to direct measurement of ACh release by EFS, and that neurotransmitters other than ACh may relate to EFS-induced contraction. In addition, it is suggested that there are prejunctional inhibitory muscarinic receptors and beta-adrenoceptors, which contribute to ACh release induced by EFS in the rabbit detrusor smooth muscles.