The present study was designed to investigate the uptake and metabolism of circulating fatty acids by the intestinal mucosa in rats actively absorbing glyceryl trioleate given intraduodenally to determine the plasma fatty acid contribution to mucosal triacylglycerol. Rats with duodenal, femoral vein, carotid artery, and mesenteric lymph duct cannulas were used. [3H]oleate was constantly infused into the femoral vein while glyceryl trioleate was infused into the duodenum (135 mumol/h). After 5 h of infusion, a mass and radioactive steady state existed in the plasma and mucosa. At 6 h of infusion, the plasma oleate specific activity was sixfold greater than mucosal oleate and 50 times greater than mucosal triacylglycerol oleate; 86% of the mucosal oleate disintegrations/minute were in triacylglycerol. Chylomicron triacylglycerol oleate specific activity was less than that of the mucosa. Furthermore, the percentage of mucosal triacylglycerol acyl groups composed of oleate was greater than the percentage of oleate in mucosal free fatty acids. The data indicate that fatty acids are taken up by the mucosa during active fat absorption and metabolized primarily to triacylglycerols by the mucosa. The triacylglycerols in the mucosa synthesized from circulating fatty acids are selected against as a precursor of chylomicron triacylglycerol. The results support our previous hypothesis suggesting that the mucosa has at least two pools of neutral lipid (J. Lipid Res. 23: 1009-1019, 1982) and that steady-state conditions as performed here yield different results from previous work using bolus tracer injection techniques.