Interstitial adenosine concentrations in red soleus (SL) or white extensor digitorum longus (EDL) muscles of anaesthetised rats were determined using microdialysis and HPLC. Systemic hypoxia was induced by ventilating the animals with 10 % oxygen in nitrogen for 15 min: arterial PO2 decreased from 111.8 ± 10.9 to 42.2 ± 4.3 mmHg (n = 4; P < 0.01) and mean systemic arterial blood pressure from 97.6 ± 4.9 to 59.0 ± 3.6 mmHg (n = 22; P < 0.001). The interstitial adenosine concentration was not significantly changed from its control values of 294 ± 44 nM (n = 20) in EDL and 302 ± 36 nM (n = 20) in SL during hypoxia or the recovery period. The interstitial lactate concentration did not change in the early part of the hypoxia but increased from 1.0 ± 0.2 to 1.4 ± 0.3 mM (n = 6; P < 0.05) in SL and from 2.0 ± 0.4 to 2.4 ± 0.4 mM (n = 6; P < 0.05) in EDL during the later part of the hypoxia, and remained elevated in the recovery period. Muscle contractions (2 Hz for 15 min) produced a transient increase in the interstitial adenosine concentration of SL from 150 ± 35 to 244 ± 75 nM (n = 10; P < 0.05) during the first 5 min of stimulation. In EDL the interstitial adenosine concentration increased from 145 ± 50 to 435 ± 144 nM (n = 10; P < 0.05) in the later part of the contraction and remained elevated in the early part of the recovery period. These data suggest that: (i) in systemic hypoxia adenosine does not appear in the interstitial space, which rules out its release from skeletal muscle, although it may be formed by the vascular tissues in this condition; (ii) adenosine is formed in the interstitial space of skeletal muscle during muscle contractions; (iii) there is slow clearance of adenosine from the interstitial space of white muscle, perhaps due to the low vascularity of the tissue. Experimental Physiology (2001) 86.5, 593-598.