The ability of proximal convoluted tubules in primary culture to regulate volume after a hypotonic shock was investigated by a method based on the use of a fluorescent intracellular probe, (2,7-bis(carboxyethyl)-5,6-carboxyfluorescein: BCECF/AM). The fluorescent signal emitted by the trapped dye excited at 450 nm and analyzed by a video-microscopic set was used to measure the relative volume change. At this wavelength the pH indicator, BCECF, was pH-insensitive and the fluorescent signal related only to the intracellular dye concentration and reflected the variations of the cellular volume as calculated from calibration data. We first determined the fading characteristics of the probe. Second, we characterized the mechanism of regulatory volume decrease (RVD) in primary cultures. RVD occurred 1 min after hypotonic shock and was complete by 4 min. This process was blocked in the presence of barium and scorpion venom (Leiurus quinquestriatus Hebraeus). In the same way, lack of chloride in external medium inhibited RVD. The Cl- blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) at 1.10(-5) M also blocked the regulation. We conclude that RVD in primary cultures of rabbit proximal convoluted tubules involves the stimulation of a potassium conductance via the Ca2(+)-activated maxi K+ channel and that the accompanying anion is chloride via a conductive pathway and (or) a KCl cotransport.