Primary cultures of pavement cells from the gills of a freshwater fish, the rainbow trout Oncorhynchus mykiss, have been studied for the first time using the patch-clamp technique. Gigaohm seals were obtained in approximately 95 % of cells studied, and channel activity was evident in a high proportion (>90 %). A large-conductance Cl− channel was evident in 6 % of cell-attached and in 31 % of inside-out patches. Single-channel conductance in inside-out patches was 372 pS, and current/voltage relationships were linear over the range −60 to +60 mV. The channel was activated by patch excision, and activation was often associated with polarization of the patch. The mean number of channels per patch was 1.9, and there were several subconductance states. The relationship between channel activity (NPo) and voltage was in the form of an inverted U, and channel activity was highest between 0 and +20 mV. Large-conductance Cl− channels showed a progressive time-dependent reduction in current in response to sustained polarization to voltages outside the range −20 mV to +20 mV. Permeability ratios (P) of Cl− to other anions were and Pisethionate/PCl=0.53. The channel was blocked by Zn2+, SITS, DIDS and diphenylamine carboxylate. This is the first description of a large-conductance Cl− channel in gill cells from freshwater or marine species. Possible functions of the channel are discussed.