ATP is an important extracellular signalling molecule which contributes to exercise vasodilation. Both acidosis and muscle contraction stimulate ATP release from skeletal muscle cells through a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent mechanism 1,2. However, it is unclear whether ATP is released through CFTR itself or whether CFTR regulates a separate ATP-release channel. We investigated whether connexin (Cx) hemichannels were involved in acidosis- or exercise-induced ATP release from rat skeletal muscle. RT-PCR indicated that cultured rat L6 skeletal myocytes expressed mRNA for both Cx40 and Cx43, but Cx40 was expressed only weakly in western blot, whereas Cx43 was strongly expressed (n=3). CFTR was isolated from a homogenate of L6 cells using an antibody bound to magnetic dynabeads. The immunoprecipitate was shown to contain both CFTR and Cx43 using western blot (n=4). In rats anaesthetised with sodium pentobarbital (70 mg/kg i.p.), gastrocnemius muscle contractions were induced by sciatic nerve stimulation, and interstitial fluid was sampled using microdialysis. In the absence of drugs, the muscle contracted with an initial force of 0.60 ± 0.05 N (mean ± SEM, n=5) and the interstitial ATP increased from 3.7 ± 0.8 to 119.6 ± 24.5 nM (P<0.02, ANOVA): both of these values were reproducible in repeated contractions. The specific CFTR inhibitor, CFTRinh-172 (20 μM) abolished the contraction-induced increase in interstitial ATP without changing the contractile force. The Cx inhibitor, gadolinium (100 μM) abolished the contraction-induced increase in interstitial ATP, but the force was also reduced from 0.68 ± 0.04 to 0.47 ± 0.04 N (n=5). However, another Cx inhibitor, carbenoxolone (100 μM) also abolished the contraction-induced increase in interstitial ATP, and did not significantly alter the contractile force, suggesting that Cx hemichannels may be involved in the contraction-induced ATP release. A Cx43 over-expression model was created by transfecting the L6 myocytes with a Cx43 plasmid using the Qiagen HiPerFect transfection medium: Cx43 over-expression was confirmed using western blot. In control myocytes, reducing the medium pH from 7.4 to 6.8 increased the extracellular ATP from 0.7 ± 0.07 to 1.2 ± 0.09 nM (n=6; P<0.001, t-test). Cx43 over-expressing myocytes released significantly more ATP than control myocytes at pH 7.4, and reducing the pH from 7.4 to 6.8 increased the extracellular ATP from 1.3 ± 0.1 to 2.1 ± 0.2 nM, suggesting that Cx43 may be involved in acidosis-induced ATP release. These data suggest that Cx43 co-localises with CFTR in the skeletal muscle cell membrane, and that a connexin may be involved in the release of ATP from skeletal muscle cells during acidosis or muscle contractions; further investigation is required to determine whether and how CFTR interacts with a Cx to induce ATP release.