Little is known about vascular effects of testosterone. We previously reported chronic testosterone treatment increases vascular tone in middle cerebral arteries (MCA; 300 microm diameter) of male rats. In the present study, we investigated the hypothesis that physiological levels of circulating testosterone affect endothelial factors that modulate cerebrovascular reactivity. Small branches of MCA (150 microm diameter) were isolated from orchiectomized (ORX) and testosterone-treated (ORX+T) rats. Intraluminal diameters were recorded after step changes in intraluminal pressure (20-100 Torr) in the absence or presence of N(G)-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor; indomethacin, a cyclooxygenase (COX) inhibitor; and/or apamin and charybdotoxin (CTX); and K(Ca) channel blockers used to inhibit endothelium-derived hyperpolarizing factors (EDHF). At intraluminal pressures >or=60 Torr, arteries from ORX+T developed greater tone compared with ORX arteries. This difference was abolished by removal of the endothelium but remained after treatment of intact arteries with indomethacin or L-NAME. In addition, testosterone treatment had no effect on cerebrovascular production of endothelin-1 or prostacyclin nor did it alter protein levels of endothelial NOS or COX-1. Endothelium removal after L-NAME/indomethacin exposure caused an additional increase in tone. Interestingly, the latter effect was smaller in arteries from ORX+T, suggesting testosterone affects endothelial vasodilators that are independent of NOS and COX. Apamin/CTX, in the presence of L-NAME/indomethacin, abolished the difference in tone between ORX and ORX+T and resulted in vessel diameters similar to those of endothelium-denuded preparations. In conclusion, testosterone may modulate vascular tone in cerebral arteries by suppressing EDHF.