The Cl- conductance of endoplasmic reticulum-enriched pancreatic microsomes was identified. Its regulation by nucleotides was investigated by measuring the rate of cation ionophore-induced microsome swelling in the presence of an inward Cl- gradient. The conductance was solubilized and reconstituted into liposomes. The Cl- conductance in intact microsomes was inhibited by stilbene (10(-4) M) and indanyloxyacetic acid (10(-5) M) derivatives. ATP increased Cl- conductance with half-maximal stimulation at 8 x 10(-6) M. Other trinucleotides (GTP, CTP and UTP) were without effect at 10(-4) M. The non-hydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate (AppCH2p) increased Cl- conductance with a potency similar to that of ATP. The same concentration of adenosine 5'-[gamma-thio]triphosphate (ATP gamma S) which is a substrate for kinases, had no effect. ATP stimulation of Cl- conductance was inhibited by stilbene derivatives. The data suggest the presence of at least one ATP-binding site, and show that the ATP does not need to be hydrolyzed and that its spatial conformation is important for activating the Cl- conductance. Solubilized microsomal proteins reconstituted into liposomes retained their stilbene-inhibited, ATP-stimulated Cl- conductance. A 167 kDa protein was detected by anti-CFTR antibodies in the intact microsomes, but not in the solubilized proteins. The 64 kDa protein (a component of a ubiquitous Cl- channel) was detected in the both intact and solubilized microsomes. These results suggest that this Cl- conductance is not a CFTR protein.