T he nonionic detergent, Pluronic L-81 (L-81) has been shown to block the transport of intestinal mucosal triacylglycerol (TG) in chylomicrons. This results in large lipid masses within the enterocyte that are greater in diameter than chylomicrons. On removal of L-81, mucosal TG is rapidly mobilized and appears in the lymph. We questioned whether the blocked TG requires partial or complete hydrolysis before its transport. Rats were infused intraduodenally with [3H]glyceryl, [14C]oleoyl trioleate (TO) and 0.5 mg L-81/h for 8 h, followed by 120 mumol/h linoleate for 18 h. Mesenteric lymph was collected and analyzed for TG content and radioactivity. An HPLC method was developed to separate TG on the basis of its acyl group species. The assumed acyl group composition was confirmed by gas liquid chromatography analysis. TG lymphatic output was low for the first 8 h but increased to 52 mumol/h at the 11th h of infusion (3 h after stopping L-81). 38% of the infused TO was retained in the mucosa after the 8-h infusion. 95% of mucosal TG was TO, 92% of the radioactivity was in TG, and 2.4% of the 14C disintegrations per minute was in fatty acid. HPLC analysis of lymph at 6, 10, 12, and 14.5 h of infusion showed a progressive rise in TG composed of one linoleate and two oleates, to 39%; and in TG composed of two linoleates and one oleate to 20% at 14.5 h of infusion. On a mass basis, however, 80% of the TG acyl groups were oleate. 3H/14C ratios in the various TG acyl group species reflected the decrease in oleate. We conclude that first, unlike liver, most mucosal TG is not hydrolyzed before transport. The mechanism of how the large lipid masses present in mucosal cells after L-81 infusion are converted to the much smaller chylomicrons is unknown. Second, the concomitant infusion of linoleate did not impair lymph TG delivery after L-81 blockade.