The isolated, short-circuited opercular epithelium of Fundulus heteroclitus, secretes Cl- by a mechanism dependent on the presence of serosal Na+ and inhibited by bumetanide and furosemide. Under serosal Na+-free conditions the active Cl- secretion is abolished. However, subsequent elevations of intracellular cyclic AMP (cAMP) levels with isoproterenol or forskolin stimulated Cl- secretion markedly. This stimulation was unaffected by SITS, DIDS, methazolamide, and HCO-3-free solutions, but was blocked by furosemide and bumetanide. Determinations of relative intracellular 36Cl- levels showed a Na+ dependence of intracellular 36Cl- in epithelia not stimulated by isoproterenol and a Na+ independence of intracellular 36Cl- in isoproterenol stimulated epithelia. In both conditions, the intracellular 36Cl- was bumetanide sensitive. The results indicate that cAMP stimulation of Cl- secretion can occur by a Na+-independent, loop diuretic-inhibitable mechanism, which may be operative even in the presence of Na+. Whether this is a separate Cl- uptake mechanism or a cAMP-induced alteration in the normal Na+-dependent mechanism could not be determined. In either instance, an alternative to the Na+ gradient as a source of energy for Cl- uptake into the cell across the basolateral membrane is required.