Primary cultures of sea bass gill cells grown on permeable membranes form a confluent, polarized, functional tight epithelium as characterized by electron microscopy and electrophysiological and ion transport studies. Cultured with normal fetal bovine serum (FBS) and mounted in an Ussing chamber, the epithelium presents a small short-circuit current (Isc: 1.4 +/- 0.3 microA/cm2), a transepithelial voltage (Vt) of 12.7 +/- 2.7 mV (serosal positive) and a high transepithelial resistance (Rt: 12302 +/- 2477 omega x cm2). A higher degree of differentiation and increased ion transport capacities are observed with cells cultured with sea bass serum: numerous, organized microridges characteristic of respiratory cells are present on the apical cell surface and there are increased Isc (11.9 +/- 2.5 microA/cm2) and Vt (25.9 +/- 1.7 mV) and reduced Rt (4271 +/- 568 omega x cm2) as compared with FBS-treated cells. Apical amiloride addition (up to 100 microM) had no effect on Isc. The Isc, correlated with an active Cl- secretion measured as the difference between 36Cl- unidirectional fluxes, was partly blocked by serosal ouabain, bumetanide, DIDS or apical DPC or NPPB and stimulated by serosal dB-cAMP. It is concluded that the chloride secretion is mediated by a Na+/K+/2Cl- cotransport and a Cl-/HCO3- exchanger both responsible for Cl- entry through the basolateral membrane and by apical cAMP-sensitive Cl- channels. This study gives evidence of a functional, highly differentiated epithelium in cultures composed of fish gill respiratorylike cells, which could provide a useful preparation for studies on ion transport and their regulation. Furthermore, the chloride secretion through these cultures of respiratorylike cells makes it necessary to reconsider the previously accepted sea water model in which the chloride cells are given the unique role of ion transport through fish gills.