To facilitate analysis of the regulation of epithelial Cl- transport by hormones, neurotransmitters, and autocrine mediators, we have developed a primary monolayer culture system for shark rectal gland (SRG) epithelial cells. Cultures exhibit vigorous transcellular Cl- secretion which can be measured using short-circuit current or 36Cl flux methods. Transport is markedly reduced by bumetanide or barium, inhibitors of Na(+)-K(+)-2Cl- cotransport and K+ channels, respectively. This indicates that Cl- secretion by SRG monolayers occurs by a mechanism similar to that described in numerous native Cl- secretory epithelia. Forskolin, 10 microM 2-chloroadenosine, or vasoactive intestinal peptide, potent secretagogues in the isolated perfused SRG, stimulate Cl- secretion by SRG cultures. Submicromolar concentrations of 2-chloroadenosine, which inhibit Cl- secretion in the native SRG, reduce forskolin-stimulated short-circuit current in SRG cultures. Somatostatin, another inhibitor of Cl- secretion by the native SRG, reduces forskolin-stimulated adenosine 3',5'-cyclic monophosphate accumulation in SRG cultures. These results demonstrate that SRG cultures are fully responsive to mediators which activate or inhibit secretion by the native epithelium.