ABSTRACTRegulatory volume decrease (RVD) following hypo-osmotic stimulation was studied using videometric methods in isolated proximal renal tubules from trout (Salmo trutta). The relative tubule diameter increased by 132.0±4.8 % (maximum swelling within 1 min at 15 and 25 °C and within 4 min at 10 °C) following a change from iso-osmotic (290 mosmol kg−1) to hypo-osmotic (160 mosmol kg−1) Ringer’s solution. The tubule diameter subsequently decreased to approximately one-quarter of the maximal value. Ouabain (1 mmol l−1) reduced cell swelling and inhibited the RVD response by 28.0±10.5 %. Furthermore, increasing the bath K+ concentration by 30 mmol l−1 inhibited RVD by 76.5±3.6 %. The K+ channel blocker quinine, but not Ba2+ (1 and 2 mmol l−1), significantly decreased the RVD response (by 25.0±5.4 and 72.3±5.1 % at 0.1 and 0.5 mmol l−1, respectively). Similarly, increasing the Cl− concentration in the bath from 47 to 102 mmol l−1 induced a significant reduction (45.2±7.9 %) in RVD. The RVD response was also markedly reduced (by 54.7±5.3 %) by the Cl− channel blocker indacrinone (MK-196; 0.5 mmol l−1), but only marginally by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 1, 5, 8 and 10 µmol l−1). Addition of the K+/Cl− symport inhibitor furosemide (0.1 mmol l−1) resulted in a 39.8±3.9 % inhibition of RVD. This inhibition could be completely overcome by simultaneous administration of 1 µmol l−1 tributyltin (anion exchanger) and furosemide.Chelation of either extracellular (1 mmol l−1 EGTA) or both extra- and intracellular Ca2+ (1 mmol l−1 EGTA, 10 µmol l−1 A23187) had no effect on this RVD process. Furthermore, as measured using the fluorescent dye Fura-2/AM, there was no increase in the intracellular free Ca2+ concentration upon hypo-osmotic stimulation. Administration of the 5-lipoxygenase antagonist ETH 615-139 (20 µmol l−1), however, induced a 60 % inhibition of RVD. Simultaneous addition of ETH-615 and either the K+ ionophore gramicidin (0.5 mmol l−1) or the anion exchanger tributyltin (1 µmol l−1) could not reverse the ETH 615-139 inhibition. Finally, administration of the cycloxygenase inhibitor indomethacin had only a small, but significant, effect on RVD.We conclude that RVD following hypo-osmotic swelling is in these cells a temperature- and ouabain-sensitive process that appears to be the result of K+ efflux through quinine-sensitive, Ba2+-insensitive K+ channels and Cl− efflux through an MK-196- and furosemide-sensitive Cl− conductance that is relatively unaffected by NPPB. This KCl efflux seems to be regulated by eicosanoids produced by the 5-lipoxygenase. Arachidonic acid metabolites from the cycloxygenase pathway are not involved in this process. Similarly, neither extra-nor intracellular Ca2+ appears to be important for the signalling of RVD.