Previous work has shown that muscle contraction elevates interstitial adenosine triphosphate concentration ([ATP]i), which is likely due to the release of ATP from active skeletal muscle. ATP activation of purinergic receptors P2X on thin muscle afferent fibers further enhances cardiovascular responses to contraction. Thus, the purposes of this study were: (1) to examine the mechanisms by which ATP is released from muscle in response to mechanical stimulation; and (2) to study the effects of interstitial ATP concentrations on modulating pressor response to muscle contraction. Static contraction of the triceps surae muscle was evoked by electrical stimulation (at 5 HZ and 2.5 times motor threshold) of the tibial nerve in 9 anesthetized cats. Muscle interstitial ATP samples were collected from microdialysis probes inserted into the muscles. Dialysate ATP concentrations were determined using the luciferin-luciferase assay. In a control experiment, contraction was induced after 0.5 ml of saline was injected into the arterial blood supply of the hindlimb muscles. This increased [ATP]i by 220% (P < 0.05 vs. baseline). After gadolinium (1 mM), a blocker of mechanically sensitive channels, was injected into the muscles, contraction increased [ATP]i by 112% (P < 0.05 vs. control). In contrast, glibenclamide (an inhibitor of the ATP-binding cassette protein), monensin, and brefeldin A, which interfere with vesicular formation (or trafficking) and inhibit exocytosis, did not significantly affect ATP release by muscle contraction. In addition, a regression analysis showed that [ATP]i was linearly related to the pressor response to muscle contraction. The data suggest that ATP release from skeletal muscle is mediated via involvement of mechanosensitive channels. These findings further support a physiological role for release of ATP in modulating cardiovascular responses during static muscle contraction.