The cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the acidosis-induced ATP release from skeletal muscle. In many cells, CFTR is activated through the cAMP/protein kinase a (PKA) pathway. We investigated whether this pathway was responsible for CFTR activation in muscle at low pH, and how the signalling pathway was initiated. Rats were anaesthetised with sodium pentobarbital (70 mg/kg i.p.); the hindquarters were perfused at 1.5 ml/min with modified Krebs Henseleit buffer (pH 7.4) and EDL muscle interstitial fluid was sampled using microdialysis. Addition of lactic acid to the perfusion medium dose-dependently elevated interstitial ATP of buffer-perfused muscle from 38 ± 8 nM in the absence to 67 ± 11 nM in the presence of 10 mM lactic acid: this increase in ATP release was abolished by the specific CFTR inhibitor, CFTRinh-172 or by the PKA inhibitor, KT5720. Forskolin increased the intersitial ATP from 99 ± 20 to 142 ± 21 nM. Addition of 10 mM lactic acid to the bathing medium of L6 skeletal myocytes increased the intracellular cAMP from 3.2 ± 0.3 to 7.1 ± 1.0 nM, the phosphor-cAMP response element-binding protein (pCREB): β-actin ratio from 0.30 ± 0.02 to 0.71 ± 0.07 and the extracellular ATP from 0.7 ± 0.1 to 1.4 ± 0.2 nM. Addition of the phosphodiesterase inhibitor, IBMX increased extracellular ATP to 3.4 ± 0.7 nM in the absence, or 5.9 ± 1.1 nM in the presence of lactic acid. Addition of 20 µM forskolin the the bathing medium increased extracellular ATP from 0.5 ± 0.02 to 1.3 ± 0.05 nM; CFTR phosphorylation was increased by the addition of forskolin alone, and further increased by the addition of forskolin plus dibutyryl-cAMP and IBMX, but the forskolin-induced increase in CFTR phosphorylation was inhibited by KT5720. Addition of the Na+/H+-exchanger (NHE) inhibitor, amiloride abolished the lactic-acid-induced increases in both the intracellular cAMP (4.2 ± 0.4 nM in the absence or 3.8 ± 0.4 nM in the presence of lactic acid) and extracellular ATP (0.8 ± 0.2 nM in the absence or 0.9 ± 0.1 nM in the presence of lactic acid). Similarly, the Na+/Ca2+-exchanger (NCX) inhibitor, SN6 abolished the lactic-acid-induced increases in both intracellular cAMP and ATP release from the myocytes. These data suggest that skeletal muscle CFTR is activated through the cAMP/PKA pathway at low pH; NHE and NCX may be involved in the signal transduction pathway linking the decreased pH to the initiation of this mechanism.