Background information . ATP is released from many cell types exposed to hypo‐osmotic shock and is involved in RVD (regulatory volume decrease). Purinergic signalling events have been extensively investigated in mammals, but not in marine teleosteans. Results . The effect of hypo‐osmotic shock on ATP release was examined in isolated hepatocytes from turbot ( Scophthalmus maximus ), a marine flatfish. Hypo‐osmotic stress (240 mOsm·kg −1 ) induced a significant increase in ATP efflux, and was inhibited by a potential CFTR (cystic fibrosis transmembrane conductance regulator) inhibitor, glibenclamide, but not by the MDR1 (multidrug resistance 1) P‐glycoprotein inhibitor, verapamil. ATP efflux could be a cAMP‐dependent process, as IBMX (isobutylmethylxanthine) and forskolin triggered the process under iso‐osmotic conditions. Protein kinases, including protein kinase C, could also be involved, as staurosporine and chelerythrine inhibited the mechanism. Calcium could contribute to ATP efflux as ionomycin, a calcium ionophore, elicited a rapid release under iso‐osmotic conditions, and chelation using EGTA abolished ATP release under hypo‐osmotic conditions. RVD was partially abolished by apyrase, an ATP scavenger, and suramin, a purinoceptor antagonist. Moreover, hypo‐osmotic shock induced a rise in intracellular calcium which could be involved in RVD. Since extracellular ATP triggered an increase in cellular free‐calcium content under iso‐osmotic conditions, our results could indicate that hypo‐osmotic‐induced ATP efflux contributes to RVD in turbot hepatocytes by stimulating purinergic receptors, which may lead to activation of a calcium signalling pathway. Conclusions . These data provide the first evidence of volume‐sensitive ATP signalling for volume maintenance in a marine teleost fish cell type.