Carbachol-induced detrusor contractions are mainly mediated via M₃ receptor subtype and depend not only on Ca²⁺ release from the intracellular calcium stores but also on Ca²⁺ influx via L-type Ca²⁺ channels. The purpose of this study was to examine the different contributions of Ca²⁺ influx and Ca²⁺ release underlying muscarinic receptor-mediated contractions in human, porcine and murine urinary bladder. Detrusor contractions were measured in urothelium-denuded detrusor strips as responses to cumulatively increasing carbachol concentrations, release of intracellular Ca²⁺ was determined in Chinese hamster ovary cells stably transfected with human muscarinic M₃ (hM₃) receptors. In human tissue, 1 μM of the L-type Ca²⁺-channel blocker nifedipine reduced carbachol contractions to 74%, in pig to 18% and in mouse to 27% of pre-drug controls. 2-Aminoethoxyphenyl borate (2-APB, 300 μM), which impairs inositol trisphosphate (IP₃)-induced release of Ca²⁺, reduced carbachol responses in human detrusor to 60%, in pig to 35% and in mouse to 20%, whereas block of the Ca²⁺-induced Ca²⁺ release with ryanodine had no significant effect on carbachol contractions in all three species. Carbachol-induced release of intracellular Ca²⁺ in Chinese hamster ovary cells expressing muscarinic hM₃ receptors was completely prevented by 100 μM 2-APB. The direct intracellular IP₃ receptor antagonist xestospongin C (10 μM) reduced carbachol-stimulated intracellular Ca²⁺ to 41% of the control value. Blockade of ATP-dependent Ca²⁺ uptake into intracellular stores with thapsigargin was associated with a concentration-dependent increase of detrusor contraction, but limited on-top contractions with carbachol. In conclusion, carbachol-induced contractions in human, porcine and mouse detrusor depend differently on Ca²⁺ influx, since potency of nifedipine reducing muscarinic receptor-mediated detrusor contraction is lower in human bladder. On the other hand, slight species differences are also found when inhibiting IP₃-induced Ca²⁺ release and Ca²⁺ reuptake into intracellular stores. Taken together, our data show considerable species differences between human, porcine and murine detrusor regarding the relative contributions of Ca²⁺ influx and maybe also carbachol-induced Ca²⁺ release that could be of relevance when using different animal models.