Serotonin (5-HT) and norepinephrine (NE) are potent vasoconstrictors in the non-erythrocyte constant-flow perfused hindlimb at 25 degrees C. Because 5-HT inhibits and NE stimulates oxygen uptake, these agonists may exert control at different sites in the vasculature that have different metabolic requirements. In this study, it was found that hypoxia, cyanide, or azide had no significant effect on 5-HT-mediated vasoconstriction; however, in the presence of cyanide and absence of glucose, vasoconstriction was transient. NE-mediated vasoconstriction displayed two prazosin-sensitive components. At low concentrations (less than 50 nM), NE-mediated vasoconstriction was totally blocked by hypoxia, cyanide, or azide. At concentrations of NE significantly greater than 50 nM, oxygen-independent vasoconstriction was observed. In conclusion, NE and 5-HT sites are biochemically distinguishable, with 5-HT sites probably located on larger vessels and supportable by anaerobic metabolism and oxygen-dependent NE sites probably located on smaller arteries or arterioles. Supraphysiological concentrations of NE may interact with a subgroup of vascular alpha 1-adrenoceptors on larger vessels giving results similar to 5-HT, thus highlighting problems with high levels of NE in the perfused rat hindlimb.