We previously demonstrated that rat tracheal arterioles and large venules constrict in response to alpha-adrenergic agonists and dilate in response to beta-adrenergic agonists. To further investigate the vasodilatory mechanisms in these vessels, we hypothesized that the vascular tone in the tracheal microcirculation is regulated in part by endogenously released nitric oxide (NO). To test this hypothesis, rat tracheal microvessels were visualized in vivo with a video microscope. The change in diameter af adventitial arteriolar (14.5 to 42.0 microm initial diameter, n = 41) and large venular (50.0 to 100.0 microm initial diameter, n = 41) microvessels following the suffusion of vasoactive drugs was measured with video calipers. Significant constriction was observed in arterioles (to 70.0% of initial diameter) and large venules (to 76.6% of initial diameter) after 20 min of suffusion with L-NAME, an inhibitor of NO synthesis. This constriction was in large part reversed by L-arginine, a biochemical precursor of NO, in arterioles (to 93.2% of initial diameter) and in venules (to 90.8% of initial diameter). The combination of prazosin, a selective alpha1-adrenergic antagonist, and yohimbine, a selective alpha2-adrenergic antagonist, also reduced L-NAME-induced constriction in arterioles (to 87.9% of initial diameter) and in venules (to 85.2% of initial diameter). L-arginine or the combination of prazosin and yohimbine alone did not affect the diameter of tracheal microvessels. These data suggest that NO exerts an important influence on tracheal microvascular tone in rats, and may attenuate alpha-adrenergic constriction in these vessels.