Previous studies suggest that collateral vessels constrict in response to serotonin. The objective of these studies was to directly measure responses of native and stimulated collateral vessels 30-400 microns in diameter to serotonin and to determine the mechanisms involved in responses to serotonin. Serotonin was suffused onto the left ventricle of dogs, and microvascular diameters were measured with computer-controlled stroboscopic illumination coupled to a microscope-video system. Stimulated collateral vessels and arterioles in collateral-dependent myocardium were measured after Ameroid constriction of the left circumflex artery. Noncollateral and native collateral vessels were examined in dogs without a constrictor. Serotonin produced dose-dependent dilation of noncollateral vessels that was decreased in native collateral vessels, stimulated collateral vessels, and vessels in collateral-dependent myocardium. Dilation in response to nitroprusside was similar in all groups. Dilation in response to serotonin was enhanced by inhibition of 5-hydroxytryptamine (5-HT)2 receptors with ketanserin and blocked by nonselective 5-HT1 and 5-HT2 receptors with methiothepin. Inhibition of nitric oxide (NO) synthase with NG-nitro-L-arginine decreased dilation in response to serotonin. Thus collaterals and arterioles in collateral-dependent myocardium are less sensitive to the dilating effect of serotonin. Responses to serotonin involve a balance between dilation mediated by 5-HT1 receptors and constriction mediated by 5-HT2 receptors.