A nonradioactive procedure using an I–-selective electrode has been developed for assaying cellular Cl– transport. NIH 3T3 fibroblasts stably transfected with the Cystic Fibrosis Trans-membrane Conductance Regulator (CFTR) Cl– channel were grown in standard 35 mm culture dishes and used to test this assay system. For efflux measurements, the fibroblasts were first incubated in an I–-loading buffer and then exposed to an I–-free buffer. Efflux was monitored using the I–-selective electrode. Application of either forskolin (5 µM) or 8-chlorphenylthio cAMP (500 µ M), to activate the CFTR Cl– channels, resulted in over a 5-fold increase in I– efflux as compared with control, untreated fibroblasts. No increase over basal efflux levels was observed in nontransfected NIH 3T3 fibroblasts treated with forskolin. The Cl– channel blockers diphenylamine-2-carboxylate (DPC) (1 mM) and 5-nitro-2-(3- phenylpropylamino)-benzoic acid (NPPB) (25 µM) reduced forskolin- stimulated efflux by 50% and 23%, respectively. In addition to forskolin, both the tyrosine kinase inhibitor genistein and the protein kinase C activator phorbol 12,13-dibutyrate, were capable of stimulating I– efflux. Thus, use of the I–-selective electrode provides a fast and convenient method for studying Cl– channels. The I– efflux assay should be useful for monitoring drug and hormone-activated Cl– transport pathways in a wide variety of cell types.