In 11 healthy volunteers, we evaluated, in a double-blind, cross-over study, whether the vasodilation that follows isometric contraction is mediated by prostaglandins PGs and/or is O₂-dependent. Subjects performed isometric handgrip for 2 minutes at 60% maximal voluntary contraction (MVC), after pre-treatment with placebo or aspirin (600mg orally), when breathing air or 40%O₂. Forearm blood flow (FBF) was measured in the dominant forearm by venous occlusion plethysmography. Arterial blood pressure (ABP) was also recorded allowing calculation of forearm vascular conductance (FVC; FBF/ABP). During air breathing, aspirin significantly reduced the increase in FVC that followed contraction at 60% MVC: from a baseline of 0.09±0.011 (mean±SEM, conductance units, CU), the peak value was reduced from 0.24±0.03 to 0.14±0.01 CU. Breathing 40% O₂, similarly reduced the increase in FVC relative to that evoked when breathing air, the peak value was 0.24±0.03 vs 0.15±0.02 CU. However, after aspirin, breathing 40% O₂ had no further effect on the contraction-evoked increase in FVC (the peak value was 0.15±0.02 vs 0.16±0.02 CU). Thus, the present study indicates that PGs make a substantial contribution to the peak of the vasodilation that follows isometric contraction of forearm muscles at 60% MVC. Given hyperoxia similarly reduced the vasodilation and attenuated the effect of aspirin, we propose the stimulus for PG synthesis and release, is hypoxia of the endothelium.