This study characterizes the distribution of various guanosine triphosphate-binding proteins (G proteins) in rat intestinal epithelial membranes. Enriched basolateral membranes were prepared from isolated enterocytes through differential density centrifugation; apical membranes were prepared with a chaotropic agent. Enrichment and purity of the membrane fractions were assessed by various biochemical markers. G proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after adenosine diphosphate ribosylation in the presence of pertussis or cholera toxins. Western blotting was performed with the use of highly specific antibodies against the following subunits: alpha Gs, alpha G1(1 or 2), alpha G 1(3), alpha Go, alpha Gz, and beta subunits. Adenosine diphosphate ribosylation catalyzed by cholera toxins revealed two major substrates of molecular weights 47 and 43 kd in only the crude and basolateral fractions. The reaction catalyzed by pertussis toxin revealed a 41 kd substrate in the crude and basolateral fractions and a 40 kd substrate in the apical fraction. Immunoblotting confirmed the presence of alpha Gs, alpha G1(1 or 2), and alpha G1(3) but failed to identify alpha Go or alpha Gz subunits in the basolateral fraction; none of these subunits were identified in the apical fraction. Beta subunits were identified in both apical and basolateral fractions. These findings suggest selective sorting of the G proteins to regional domains in the plasma membrane of intestinal epithelial cells. The presence of previously unidentified G proteins is also suggested.