Na-K-Cl cotransport stoichiometry and affinities for Na, K and Cl were determined in flounder intestine. Measurement of simultaneous NaCl and RbCl influxes resulted in ratios of 2.2 for Cl/Na and 1.8 for Cl/Rb. The effect of Na and Rb on Rb influx showed first order kinetics with K1/2 values of 5 and 4.5 mM and Hill coefficients of 0.9 and 1.2, respectively. The effect of Cl on rubidium influx showed a sigmoidal relationship with K1/2 of 20 mM and a Hill coefficient of 2.0. The effects of variations in Na and Cl concentration on short-circuit current (Isc) were also determined. The K1/2 for Na was 7 mM with a Hill coefficient of 0.9 and the K1/2 for Cl was 46 mM with a Hill coefficient of 1.9. Based on the simultaneous influx measurements, a cotransport stoichiometry of 1Na:1K:2Cl is concluded. The Hill coefficients for Cl suggest a high degree of cooperativity between Cl binding sites. Measurements of the ratio of net Na and Cl transepithelial fluxes under short-circuit conditions (using a low Na Ringer solution to minimize the passive Na flux) indicate that the Cl/Na flux ratio is approximately 2:1. Therefore, Na recycling from serosa to mucosa does not significantly contribute to the Isc. Addition of serosal ouabain (100 microM) inhibited Rb influx, indicating that Na-K-Cl cotransport is inhibited by ouabain. This finding suggests that a feedback mechanism exists between the Na-K-ATPase on the basolateral membrane and the apical Na-K-2Cl cotransporter.