Detrusor smooth muscle contraction following activation of muscarinic receptors involves both myosin light chain kinase (MLCK) and Rho kinase. Previous studies in rodents have revealed substantial species differences in the differential contribution of these enzymes, and it is not known which pathway is predominantly involved in humans. Here, we investigated the role of MLCK and Rho kinase in human detrusor smooth muscle from bladder cancer patients who underwent cystectomy. Therefore, we prepared tiny muscle stripes from the macroscopically healthy bladder wall and measured isometric contractions of these specimens in an organ bath. Following pharmacological activation of muscarinic receptors with carbachol strong contractions could be elicited that were entirely sensitive to atropine. Pre-incubation of these specimens with either the MLCK inhibitor ML-9 or the Rho kinase inhibitors HA1100 and Y-27632, respectively, significantly blocked carbachol-induced contractions as compared to time-control experiments that showed only little run-down. Interestingly, MLCK and Rho kinase inhibition were equally effective in reducing carbachol-induced contractions. Moreover, carbachol application following inhibition of both MLCK and Rho kinase led to residual contractions that were significantly less than the contractions obtained when only one enzyme was blocked (either MLCK or Rho kinase) indicating an additive effect of both kinases. Our results indicate that both MLCK and Rho kinase considerably contribute to carbachol-induced contractions, and may suggest Rho kinase inhibitors as a new pharmacological approach to modulate human bladder hyperactivity.