Our earlier study [Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G673-G679, 1989] showed that the isomers prostaglandin E2 and D2 (PGE2 and PGD2) had contrasting effects on the canine proximal colonic epithelium in vitro. Whereas PGE2 produced sharp increases in short-circuit current (Isc), PGD2 rapidly reversed these changes. We report here that PGD2 reversed increases in Isc produced by other agonists (forskolin, carbachol) and was thus not a specific antagonist to PGE2. The transient increase in Isc and conductance produced by PGE2 were accompanied by alterations in Cl- but not Na+ fluxes. These were partitioned into early and late phases. In the early phase, net Cl- flux (JCl-net) decreased due to a reduction in Cl- mucosal to serosal flux (JCl-m----s) and increases in JCl-s----m. In the later phase, these changes appeared to revert to prestimulation values. Because forskolin produced more sustained increases in Isc, we used it as an agonist to define the effects of PGD2 on ion fluxes. Forskolin produced a marked reduction in JCl-net due to a decrease in JCl-m----s and an increase in JCl-s----m. PGD2 not only reversed the increases in Isc and conductance produced but also reversed the changes in Cl- flux.