The nonhydrolyzable guanyl nucleotide GTP gamma S stimulated phosphoinositidase C activity in two preparations obtained from mouse pancreatic acini labeled with myo[2-3H]inositol: a cell-free membrane fraction and intact electropermeabilized acini. This action was dose-dependent, was shared by other nonhydrolyzable guanyl nucleotides such as GMP-phencyclidine hydrochloride and GMP-PMP, as well as by fluoride, and was calcium-independent. Contrarily, no effect was observed even at doses of GTP gamma S as high as 10 microM when the same protocol was repeated on identical acinar preparations from mice fed a choline-deficient, ethionine-supplemented diet. This regimen is known to uncouple secretagogue-receptor occupancy from inositol 1,4,5-trisphosphate generation in pancreatic acinar cells and lead to necrotizing hemorrhagic pancreatitis. These data lead us to conclude that the ethionine-induced inactivation of guanyl nucleotide-dependent pancreatic phosphoinositidase C in pancreatic acinar cells is not the result of either a decrease in GTP level or a decrease in GTP availability. These findings further confirm previous work from this laboratory, which has shown that the biochemical lesion induced by this diet occurs after the agonist-receptor binding step. The diet-induced lesion could be either at the level of the G-protein that couples the enzyme with the receptor or at the level of the phospholipase itself.