Cl- channel and Na(+)-K(+)-2Cl- cotransport activities were studied in various cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cells with the aim of assessing integrative patterns of regulation of Cl- secretion. Human colonic HT-29 cells express relatively high levels of CFTR and cotransporter but relatively low Cl- channel activity. These cells showed commensurate activations of both transport systems evoked by short-term (minutes) or long-term (hours) exposures to adenosine 3',5'-cyclic monophosphate (cAMP). However, unlike in the case of CFTR and Cl- channels, long-term induction of cotransporter did not depend on de novo protein synthesis or changes in number of transporters. The patterns of activation of both transporters were also examined in CFTR-deficient cell lines (CFPAC and the viral-transfected CFPAC-PLJ) and in the viral CFTR-transfected derivative (CFPAC-4.7). All these cells displayed relatively low basal cotransport activity and a correspondingly low number of transporters, whereas only CFPAC-4.7 cells showed short-term (but not long-term) activatable Cl- channels. However, irrespective of the presence or absence of CFTR in CFPAC cells, neither short- nor long-term cAMP exposures induced significant cotransporter activation. Our studies with the various epithelial cell lines indicate that expression of CFTR activity per se is not sufficient for stimulation of cotransporter activity. Moreover, despite apparent correction of CFTR levels in CFPAC cells by gene transfer, the apparent Cl- secretory capacity might be limited by the low cotransport activity, such as that found in CFPAC cells, with obvious implications for proposed gene therapy of cystic fibrosis.