A 120 d feeding study with adult rats was conducted to evaluate the influence of two protein sources (casein and gelatin), two protein levels (50 and 300 g/kg diet) and two fat levels (50 and 150 g/kg diet) on serum lipids (total cholesterol, HDL-cholesterol and triacylglycerols) and liver polyunsaturated fatty acid levels. In general, the concentrations of serum triacylglycerols and total cholesterol and liver phospholipid levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in rats fed on casein diets compared with those fed on the gelatin diets. These effects were more pronounced in rats fed on the high-casein (300 g/kg)-high-fat (150 g/kg) diet. Gelatin was hypocholesterolaemic and also suppressed the liver phospholipid levels of AA and DHA (reported for the first time). The difference in the amino acid composition between casein and gelatin may be responsible for the observed effects. Casein contains higher levels of glutamic acid, methionine, phenylalanine and tyrosine, while gelatin contains higher levels of arginine, glycine and hydroxyproline. It is suggested that a protein source which increases serum cholesterol may also increase the concentrations of AA and DHA in rat tissues.