The structure of mucosal triacylglycerols was studied in rat intestinal mucosa in vivo during the absorption of a low molecular weight fraction of butter oil and of the corresponding free fatty acids of medium and long chain length. The mucosal lipids were isolated by solvent extraction and the acylglycerol structures were determined by combined AgNO3 - TLC and GLC techniques and stereospecific analysis. Evidence was obtained for a rapid biosynthesis of triacylglycerols from diacylglycerols arising from the operation of both the monoacylglycerol and the phosphatidic acid biosynthetic pathways. Both sn-1,2- and sn-2,3-diacylglycerols appeared to be converted to triacylglycerols at significant rates, but a preferential utilization of sn-1,2-diacylglycerols could not be excluded. Endogenous dilution varied from a minumum of 5% during triacylglycerol biosynthesis from monoacylglycerols to 15% during their synthesis from free fatty acids, and was characterized by a preferential placement of the endogenous acids in the sn-3 and 2 positions of the triacylglycerol molecules. Exogenous myristic acid was preferentially associated with the sn-3 position, and stearic acid became preferentially bound to the sn-1 position. The complexity of the triacylglycerol end products prevented an exact estimate of the contribution of the phosphatidic acid pathway, but the acylglycerol structures were compatible with a minimum of 20% of total triacylglycerol yield at all times.