Intraluminal ATP could play an important role in the local regulation of skeletal muscle blood flow, but the stimuli that cause ATP release and the levels of plasma ATP in vessels supplying and draining human skeletal muscle remain unclear. To gain insight into the mechanisms by which ATP is released into plasma, we measured plasma [ATP] with the intravascular microdialysis technique at rest and during dynamic exercise (normoxia and hypoxia), passive exercise, thigh compressions and arterial ATP, tyramine and ACh infusion in a total of 16 healthy young men. Femoral arterial and venous [ATP] values were 109 ± 34 and 147 ± 45 nmol l(−1) at rest and increased to 363 ± 83 and 560 ± 111 nmol l(−1), respectively, during exercise (P < 0.05), whereas these values did not increase when exercise was performed with the other leg. Hypoxia increased venous plasma [ATP] at rest compared to normoxia (P < 0.05), but not during exercise. Arterial ATP infusion (≤1.8 μmol min(−1) increased arterial plasma [ATP] from 74 ± 17 to 486 ± 82 nmol l(−1) (P < 0.05), whereas it remained unchanged in the femoral vein at ∼150 nmol l(−1). Both arterial and venous plasma [ATP] decreased during acetylcholine infusion (P < 0.05). Rhythmic thigh compressions increased arterial and venous plasma [ATP] compared to baseline conditions, whereas these values did not change during passive exercise or tyramine infusion. These results demonstrate that ATP is released locally into arterial and venous plasma during exercise and during hypoxia at rest. Compression of the vascular system could contribute to the increase during exercise whereas there appears to be little ATP release in response to increased blood flow, vascular stretch or sympathetic ATP release. Furthermore, the half-life of arterially infused ATP is <1 s.