The effects of epidermal growth factor (EGF) and its interaction with dietary adaptation were examined in pancreatic acinar cells isolated from rats fed for 1 wk diets with 67% kcal as fat (HF), 67% kcal as carbohydrate (HC), or unrefined, closed formula diet (UD). Cells were cultured for 48 h in serum-free medium containing 4.2 nM (control), 42 pM, 0.42 nM or 42 nM EGF. EGF at the concentrations tested did not affect cellular protein, DNA, amylase and lipase. In freshly isolated and cultured cells, specific activity of intracellular phenylalanine was affected by diet with the highest specific activity in HF cells, but was not affected by EGF. In freshly isolated cells, EGF increased the rate of phenylalanine incorporation into cellular protein only in HF cells at 42 nM. In cultured cells, EGF biphasically increased phenylalanine incorporation at 42 pM and 42 nM in UD and HC cells, but only increased phenylalanine incorporation in HF cells at 42 nM. These data suggest that diet can alter the response of pancreatic acinar cells to EGF. Dietary alterations of cellular responsiveness to regulatory peptides may participate in pancreatic regulation and dietary adaptation in vivo.