Angiotensin (AII) and serotonin (5-HT) are both vasoconstrictors of the constant-flow perfused rat hind limb that have opposite effects on thermogenesis, possibly the result of differing effects on vascular flow distribution between nutritive and non-nutritive pathways. In the present study interaction between the two opposing agents was examined with the expectation that the combined presence would show additive effects on pressure and mutually neutralizing effects on thermogenesis. Thus doses of AII and 5-HT that gave similar, but opposite, quantitative effects on thermogenesis were infused alone, in combination one after the other, or in combination with the order reversed, and the effects on perfusion pressure (PP) and thermogenesis (oxygen uptake, VO2) were compared. AII (3 nM) alone increased PP by 15+/-1 mmHg (1 mmHg = 133.3 Pa) and VO2 by 3.1-/+0.2 micromol.h(-1).g(-1), whereas 5-HT (1 microM) alone increased PP by 75+/-6 mmHg and inhibited VO2 by 3.9+/-0.2 micromol.h(-1).g(-1). When added in combination, the outcome depended on the order of addition. Following AII, infusion of 5-HT further increased PP by 160+/-11 mmHg and decreased VO2 by 6.3+/-0.2 micromol.h(-1)g(-1). Following 5-HT, infusion of AII further increased PP by 28+/-4 mmHg and increased VO2 by only 1.8+/-0.3 micromol.h(-1).g(-1). The prior presence of 5-HT (1 microM) shifted the AII dose-response curves for VO2 and pressure to the right and left, respectively. The prior infusion of AII increased the dose-dependent response to 5-HT in terms of both the inhibition of VO2 and the increase in PP. At low doses of 5-HT (10(-8)-10(-7) M), but not alpha-methyl serotonin (alphaMT), there was a marked vasodilatation-associated inhibition of AII-mediated increase in VO2. Overall the data show that the combined effect of AII and 5-HT differed from the simple addition of each separately. Since the order of addition appears to be critical in terms of thermogenic outcome, it is concluded that each vasoconstrictor exerts a specific hemodynamic action to affect access of the other to vascular receptor sites. These findings are consistent with the previously reported effects of these vasoconstrictors on substrate and insulin access to muscle of the perfused rat hind limb.