To investigate a possible relation between hypercholesterolemia and detrusor smooth muscle function, we studied the contractile response to potassium challenge, carbachol (CCh), and the components of CCh-induced contractile mechanism in high-cholesterol diet-fed rats. Adult male Sprague-Dawley rats were fed with standard (control group, N = 17) or 4 % cholesterol diet (hypercholesterolemia group (HC), N = 16) for 4 weeks. Spontaneous contractions of detrusor muscle strips and their responses to potassium chloride (KCl) or cumulative dose-contraction curves to CCh were recorded. The effects of muscarinic receptor antagonists (methoctramin and/or 4-diphenylacetoxy-N-methylpiperidine), L-type Ca+2 channel blocker (nifedipine), and/or rho-kinase inhibitor Y-27632 were investigated. Blood cholesterol level was increased in the HC group with no sign of atherosclerosis. The KCl-induced detrusor smooth muscle contractions were higher in HC, whereas spontaneous and CCh-induced responses were similar in both groups. Preincubation with receptor antagonist for M-3 but not for M-2 attenuated contraction significantly, shifting the dose-response curve to the right. This response was similar in both groups. Among two effector mechanisms of M-3-mediated detrusor smooth muscle contraction, rho-kinase pathway was not affected by hypercholesterolemia, whereas blockade of L-type Ca+2 channels potently reduced contractions. The results of this study point out a relation between hypercholesterolemia and contractile mechanism of detrusor smooth muscle likely to change urinary bladder function, via altering L-type Ca+2 channels. Taken together with escalating incidence of hypercholesterolemia and lower urinary tract symptoms, it is a field which deserves to be investigated further.