The intestine can vary its triacylglycerol output rate depending on differing physiological conditions. The rate-limiting step in the complex process from fatty acid and monoacylglycerol entry to triacylglycerol export is unknown but suggested to be the transport of triacylglycerol from the endoplasmic reticulum to the Golgi. The present studies were carried out to test this hypothesis. The conversion rate of absorbed fatty acid to mucosal triacylglycerol was studied in rats infused intraduodenally with trioleoylglycerol, 135 micromol/h, for 6 h followed by [3H]oleate. In 30 sec, 79% of the mucosal 3H-labeled fatty acid was esterified to [3H]triacylglycerol. The increase in the 3H specific activity of triacylglycerol in the endoplasmic reticulum and Golgi was studied in similarly prepared rats except that the radio-label was [3H]trioleoylglycerol. The endoplasmic reticulum triacylglycerol specific activity was always less than that of the Golgi with a steady state not reached until 60 min of [3H]trioleoylglycerol infusion. The steady state of [3H]triacylglycerol in the lymph was not reached until 70 min of infusion. We conclude that the data are consistent with the rate-limiting step in intestinal triacylglycerol export being the movement of triacylglycerol from the endoplasmic reticulum to the Golgi as the conversion of absorbed fatty acid to triacylglycerol is rapid and the movement of triacylglycerol from the Golgi to the lymph is rapid as well.