The effects of CLCA protein expression on the regulation of chloride conductance by intracellular Ca²⁺ and cyclic AMP have been studied previously in non-epithelial cell lines chosen for low backgrounds of endogenous chloride conductance. However, CLCA proteins have been cloned from, and normally function in differentiated epithelial cells. In this study we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of chloride conductance by pCLCA1 protein expression. Chloride transport was measured as ³⁶Cl efflux, as transepithelial short circuit currents, and as whole cell patch clamp current/voltage relationships. The rate of ³⁶Cl efflux and amplitude of currents in patch clamp studies after addition of the Ca²⁺ionophore A23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1- dependent Ca²⁺-sensitive chloride conductance could be detected in 14-day post-passage cells. Unlike Ca²⁺-sensitive chloride conductance, endogenous cAMP-dependent chloride conductance does not disappear upon Caco-2 differentiation. cAMP-dependent chloride conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed both in freshly passaged, and in mature 14-day post-passage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent chloride conductance were retained through differentiation in Caco-2 cells, while calcium-dependent chloride conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous chloride channels to Ca²⁺ and cAMP agonists, but apparently lacks inherent chloride channel activity under growth conditions where endogenous channels are not expressed.