ATP is released from skeletal muscle cells under basal conditions, and at higher rates during acidosis or muscle contractions. The cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the stimulated ATP release, but neither inhibition of CFTR nor silencing of CFTR expression could abolish the basal ATP release. Chloride channels such as the maxi anion channel or voltage-sensitive-outwardly- rectifying (VSOR) channel are known to mediate ATP release in a number of cell types. We investigated whether chloride channels were involved in the release of ATP from rat skeletal muscle. Using RT-PCR (n=3), rat L6 skeletal myocytes were shown to express CFTR, ClC 2, 3 and 7, and VDAC (Voltage-dependent anion channel); the maxi anion channel and VSOR, whose molecular identities are uncertain, were not tested. Whole cell chloride currents were measured in the myocytes using patch clamp: reducing the pH from 7.4 to 6.8 increased the current from 69.8 ± 8.8 to 98.6 ± 9.6 pA/pF (mean ± SEM, n=9; P<0.05, ANOVA) at 70 mV; this increase could be partially inhibited by the specific CFTR inhibitor, CFTRinh-172. Addition of DIDS (100 μM) reduced the current to 16.2 ± 3.4 pA/pF at pH 7.4; lowering the pH to 6.8 in the presence of DIDS increased the current to 25.6 ± 3.8 pA/pF (P<0.05; n=4). Gastrocnemius muscle contractions were induced in rats anaesthetised with sodium pentobarbital (70 mg/kg i.p.) by stimulation of the sciatic nerve at 1 Hz and supramaximal voltage; the interstitial fluid was sampled using microdialysis. In the absence of drugs, muscle contractions increased the interstitial ATP from 1.4 ± 0.1 to 44.1 ± 12.8 nM (n=5); this increase was reproducible in repeated contractions in the absence of drugs. Injection of DIDS (100 μM) reduced the interstitial ATP to 0.3 ± 0.05 nM at rest and 3.8 ± 2.0 nM during contractions. During buffer-perfusion of the rat hindquarters, infusion of lactic acid (10 mM) increased the EDL muscle interstitial ATP from 31 ± 5 to 50 ± 7 nM (n=14); addition of SITS (100 μM) to the perfusate abolished the lactic-acid-induced increase in ATP. The ATP concentration in the medium surrounding the cultured L6 myocytes was increased from 0.6 ± 0.06 to 1.1 ± 0.08 nM (n=57; P<0.001, t-test) by the addition of 10 mM lactic acid to the medium. Addition of the maxi anion channel inhibitors gadolinium (100 μM, n=12) or sodium orthovanadate (100 μM, n=6) to the medium increased the extracellular ATP, but the acidosis-induced increase in ATP was abolished. The VSOR inhibitor, phloretin (100 μM, n=6) also increased extracellular ATP, but failed to inhibit a further increase in ATP when the pH was reduced to 6.8. These data suggest that lowering of the pH opens both CFTR and another chloride channel, and that a chloride channel, which may be the maxi-anion channel, contributes to ATP release from skeletal muscle.