Single cell optical imaging techniques were used to compare Cl⁻ conductances in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing and control mouse L cell fibroblasts. Elevation of intracellular cAMP levels in control cells was without effect on plasma membrane Cl⁻ permeability, whereas cells engineered to stably express CFTR displayed a 20-fold enhancement of plasma membrane Cl⁻ permeability in response to cAMP. Control L cells displayed Ca²⁺-, as well as swelling-activated Cl⁻ permeabilities, which were small compared with cAMP-stimulated permeability in CFTR-expressing cells. CFTR-expressing cells also displayed a similar swelling-activated Cl⁻ permeability. Whereas 50% of the CFTR-expressing cells possessed a small Ca²⁺-activated Cl⁻ permeability similar to control cells, the other cells displayed an enhanced response which was never observed in control cells. Intracellular cAMP determinations suggested that this latter result might be explained by a Ca²⁺-induced rise of cAMP. The cAMP-activated and Ca²⁺-activated Cl⁻ conductances had different anion selectivities, as measured by light scattering of suspended cells. Activation of protein kinase C was without effect on Cl⁻ permeability in CFTR-expressing cells, nor did it modify cAMP-activation of Cl⁻ permeability. Thus, expression of human CFTR in L cells does not confer cAMP-sensitivity to pre-existing, endogenous Ca²⁺-or swelling-activated Cl⁻ channels, but rather confers a novel Cl⁻ conductance which is regulated by cAMP. Osmotic cell swelling and PKC activation are without specific effect in CFTR-expressing L cells. However, elevated [Ca²⁺]_i may play a role in activating a Cl⁻ conductance specifically associated with CFTR.