We have previously shown great similarity in the distribution of fatty acids in the sn-1 and sn-3 positions of the chylomicron triacylglycerols (TG) from rats fed menhaden oil or its ethyl esters, and have proposed that the acylglycerol products of the phosphatidic acid (PA) pathway (ester feeding) are hydrolyzed to 2-monoacylglycerols (2-MG) prior to reconversion to TG via the 2-MG pathway (oil feeding) and secretion as chylomicrons. As the composition of the sn-2-position would also be retained if the TG were hydrolyzed only to the X-1,2-diacylglycerol (DG) stage before resynthesis, we have now retested the hypothesis by determining the molecular association and reverse isomer content of the sn-1,2- and sn-2,3-DG derived from the chylomicron TG and the PA resulting from the two feedings. The new data demonstrate a better than 90% homology among the molecular species of the PA from the oil and ester feeding, along with the characteristic association of the saturated acids with sn-1- and the unsaturated acids with sn-2-position. Due to increased proportion of unsaturated acids in the sn-1-position of the TG, there was only a 15-20% homology between the PA and the sn-1,2-DG moieties of the chylomicron TG from the oil and ester feeding. A lack of homology was also observed between the PA and free sn-1,2-DG, as well as between the free sn-1,2-DG and the sn-1,2-DG moieties of the chylomicron TG. On the basis of molecular association and the sn-1-/sn-3- reverse isomer content of the chylomicron TG a better than 90% homology was recognized between the chylomicron TG resulting from the oil and ester feeding. It is therefore concluded that hydrolysis to 2-MG followed by reesterification via the 2-MG pathway constitutes the most plausible mechanism for the transfer to chylomicrons of the TG arising from alkyl ester feeding.