The abundance of membrane cholesterol is an important determinant of the functional properties of biomembranes. To determine whether arterial smooth muscle acquires altered contractile properties in a high cholesterol environment, isolated canine coronary arteries were exposed to cholesterol in stable aqueous solution. Cholesterol, 10(-12) to 10(-10) M, was an efficacious vasoconstrictor, as maximum contractions equaled those obtained with 15 mM KCl. Antiadrenergic interventions, including chemical sympathectomy in vivo with 6-hydroxydopamine and alpha- and beta-adrenergic blockade with phentolamine and L-propranolol (both 10(-6 M), did not significantly attenuate the contractions. However, responses to cholesterol were abolished completely by (+/-)-verapamil (10(-6) M). Cholesterol in picomolar concentration enhanced the constrictor effects of CaCl2 and KCl, both in the presence and absence of alpha- and beta-adrenergic blockade. Increases in tone in response to graded elevations in the CaCl2 concentration (0-2 mM) were augmented up to 1.5-fold by 10(-12) M cholesterol (P less than 0.01). Results indicate that cholesterol sensitizes isolated coronary arteries to external Ca2+ by a nonadrenergic mechanism. The findings are consistent with the hypothesis that acquisition of membrane cholesterol may alter the contractile properties of coronary arterial smooth muscle, a phenomenon that could play a role in the pathophysiology of atherosclerotic heart disease.