The activity, synthesis rate and mRNA level of pancreatic lipase increase with dietary fat intake. Ketones, intermediates of lipid metabolism, have been proposed to mediate this change. Therefore, we investigated their direct effect on cultured pancreatic acinar cells and examined their possible interactive effects with glucose and dietary fat. beta-Hydroxybutyrate (0.01 to 2 mmol/L) did not affect lipase activity in cells isolated from rats fed a commercial nonpurified (NP) diet and cultured in high glucose (HG, 27.8 mmol/L) or low glucose (LG, 6.9 mmol/L) medium. The effects of ketones were also examined in acinar cells isolated from rats fed purified high fat (HF, 67% of energy from fat) or low fat (LF, 11% of energy from fat) diet. Cellular lipase was significantly higher in cells from HF-fed rats at both 24 and 48 h (264% and 145% of LF values, respectively; P less than 0.0001). beta-Hydroxybutyrate significantly increased (P less than 0.04) lipase activity in LF cells at 48 h but did not affect lipase activity in HF cells. These studies suggest that ketones may be involved in the regulation of pancreatic lipase in rats fed a LF diet, but their role is complex and interactive with dietary carbohydrate and fat.