At rest, systemic hypoxia induces vasodilatation in skeletal muscle that is largely attributable to adenosine. Our in vivo and in vitro studies showed that in hypoxia, adenosine acts on A1 receptors on endothelial cells, generating PGI2 as an intermediate. PGI2 acts back on endothelial cells to generate cAMP, increase nitric oxide synthase (NOS) activity and release NO, which causes vasodilatation (Ray et al, 2002). The hypoxia-induced release of adenosine is also NO-dependent. Thus, providing a tonic level of NO is present, endothelial cells in vivo or in vitro, release adenosine in response to NO donor or hypoxia. We deduced this reflects the competitive interaction between NO and O2 at their binding site on cytochrome oxidase (cyta3). Tonic NO raises the sensitivity of cyta3 to O2 such that even O2 levels reached in systemic hypoxia, decrease endothelial ATP synthesis leading to adenosine release (see Edmunds et al, 2003; Ray & Marshall, 2005). The question arises as to whether these same mechanisms operate in exercise, for adenosine, prostaglandins (PG) and NO are implicated in exercise hyperaemia. Our results suggest not. In anesthetized rats, hyperaemia associated with tetanic or twitch contractions for 5 minutes was partly mediated by adenosine, but acting via A2A receptors, not A1 receptors. Further, the contribution of adenosine to exercise hyperaemia persisted after NOS inhibition: neither the release nor action of adenosine is NO-dependent. Since systemic hypoxia increases adenosine in plasma whereas exercise increases adenosine in interstitium, the simplest explanation is that in exercise, adenosine mainly acts on extralumininal A2A receptors on vascular smooth muscle to directly cause dilatation Ray & Marshall, 2009a,b). Nevertheless, our findings indicate that exercise is at least partially dependent on a fall in O2 levels, but in the vicinity of skeletal muscle fibres rather than in plasma. The hyperaemia following static forearm contraction at 50% MVC for 3 minutes was similarly decreased by breathing 40% O2 before, during and after contraction, or by blockade of cyclooxygenase (COX) to inhibit PG synthesis. Further, supplementary O2 and COX applied together had no greater effect than either independently (Win & Marshall, 2005). We recently showed that breathing 40% O2 attenuated post-contraction hyperaemia when given during static contraction at 100%MVC, but not when given throughout recovery. Also, supplementary O2 during contraction decreased venous efflux of lactate and hydrogen ions. This indicates that increasing the level of O2 in plasma increase the partial pressure of O2 in muscle fibres and so affects their metabolism (Fordy & Marshall, 2012). In our most recent studies, 40% O2 restricted to a 2 min period of static at 50% maximum voluntary contraction (MVC) or to rhythmic contraction at 50% MVC at 1sec intervals, reduced the post-contraction by ~30-40%; COX inhibition or both applied together had similar effects. Thus, the contribution of PGs to the hyperaemia associated with even modest levels of contraction, that are not generally expected to involve impaired O2 delivery, is apparently dependent in some way on a fall in O2 levels in muscle. We have followed this up in studies on the rat, in which highly selective adenosine receptor antagonists can be used: theophylline that is available for use in humans is a relatively weak competitive antagonist of adenosine receptors. So far, we have found that breathing 40% O2 or 8-suphophenyltheophylline (8-SPT) similarly decreased hyperaemia associated with tetanic contraction and that 40% O2 and 8-SPT applied together had no greater effect. Drawing these results together, we propose that during exercise, adenosine is released from skeletal muscle into interstitium by an O2-dependent process and that it acts on extraluminal A2A receptors on arterioles to cause exercise hyperaemia. A contribution is also made by PGs that are released from skeletal muscle fibres by an O2-dependent process. Whether these O2-dependent influences are interdependent remains to be investigated, but it seems unlikely that adenosine acts on A1 receptors to generate PGI2 via the mechanisms we implicated in systemic hypoxia.