Previous studies performed on apical membranes of seawater fish gills in primary culture have demonstrated the existence of stretch-activated K⁺ channels with a conductance of 122 pS. The present report examines the involvement of K⁺ channels in ion transport mechanisms and cell swelling. In the whole cell patch-clamp configuration, K⁺ currents were produced by exposing cells to a hypotonic solution or to 1 µM ionomycin. These K⁺ currents were inhibited by the addition of quinidine and charybdotoxin to the bath solution. Isotopic efflux measurements were performed on cells grown on permeable supports using ⁸⁶Rb⁺ as a tracer to indicate potassium movements. Apical and basolateral membrane ⁸⁶Rb effluxes were stimulated by the exposure of cells to a hypotonic medium. During the hypotonic shock, the stimulation of ⁸⁶Rb efflux on the apical side of the monolayer was inhibited by 500 µM quinidine or 100 µM gadolinium but was insensitive to scorpion venom [Leirus quinquestriatus hebraeus (LQH)]. An increased ⁸⁶Rb efflux across the basolateral membrane was also reduced by the addition of quinidine and LQH venom but was not modified by gadolinium. Moreover, basolateral and apical membrane ⁸⁶Rb effluxes were not modified by bumetanide or thapsigargin. There is convincing evidence for two different populations of K⁺ channels activated by hypotonic shock. These populations can be separated according to their cellular localization (apical or basolateral membrane) and as a function of their kinetic behavior and pharmacology.