The effects of propionate, a product of intestinal fiber fermentation, on fatty acid and sterol synthesis were studied in isolated rat hepatocytes. Fatty acid synthesis, as measured by tritium incorporation from 3H2O, was inhibited in the presence of 1 mmol/L propionate with no substrate additions or additions of acetate, butyrate, lactate or oleate. Incorporation of [1-14C]acetate into fatty acids was also inhibited in the presence of propionate. Although propionate markedly depressed [1-14C]acetate incorporation into sterols in hepatocyte preparations, tritium incorporation from 3H2O into sterols was not inhibited, indicating that overall sterol synthesis was not affected. Thus, in vitro, the effect of propionate on lipid metabolism is apparently limited to inhibition of de novo fatty acid synthesis.