1. Rates of in vivo fatty acid (FA) synthesis were assessed with 3H2O in carcass, liver, intestine, muscle and four adipose depots from rats fed a high-protein, carbohydrate-free diet (70% casein, 8% fat, w/w) or a balanced diet (66% carbohydrate, 17% casein, 8% fat) for 25-30 days. 2. Rats adapted to the high protein (HP) diet showed a marked reduction of total FA synthesis from all carbon sources, which was due to a decreased synthesis of triacylglycerols. Rates of phospholipid-fatty acid synthesis in both carcass and liver were not affected by the diet. Rates of triacylglycerol-fatty acid (TAG-FA) synthesis were markedly reduced in adipose tissue from four different sites: epididymal (79%), retroperitoneal (78%), subcutaneous (65%) and intermuscular (82%). 3. In rats fed the balanced control diet, TAG-FA synthesis in adipose tissue accounted for about 75% of synthesis in whole carcass, whereas it was reduced to 36% in rats under the HP regimen. 4. Although hepatic lipogenesis was also reduced in HP-fed rats, the contribution of the liver to total TAG-FA synthesis was approximately the same in HP (24%) and control (20%) rats, whereas the contribution of adipose tissue was only 26% in HP-fed animals compared to 57% in controls. 5. Force-feeding fed rats with components of their own diets resulted in a significant (100%) increase of liver TAG-FA synthesis in animals fed the control diet, but did not significantly affect liver lipogenesis in HP rats.