Endometrial platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) levels change significantly during the pre-implantation period in the rabbit uterus, but under in vitro culture conditions, constitutive PAF biosynthesis by isolated endometrial tissues is not easily demonstrable. Rapid metabolism of PAF relative to its synthesis may account for this disparity because we have recently shown that in stromal cells there is a significant build-up of lyso-PAF suggesting that lyso-PAF-acetyl-CoA acetyltransferase might be a limiting factor. In the glandular epithelial cells however, the lyso-PAF build-up was replaced by a significant accumulation of a neutral lipid which was tentatively identified as 1-O-hexadecyl-2-acetylglycerol. It was hypothesized that, during endometrial growth and development, this lipid might serve as the substrate for the alkylacetylglycerol CDP-choline cholinephosphotransferase enzyme for PAF synthesis via the de novo pathway. We have therefore examined the activities of lyso-PAF:acetyl-CoA acetyltransferase and the CDP-cholinephosphotransferase enzymes. Microsomal preparations containing lyso-PAF:acetyl-CoA acetyltransferase activity catalyzed the incorporation of [3H]acetyl-CoA lyso-PAF into two distinct lipid products. One co-migrated with authentic PAF and the other with 1-O-hexadecyl-2-acetylglycerol, the latter being formed subsequent to PAF formation. The alkylacetylglycerol CDP-choline cholinephosphotransferase enzyme, which would potentially utilize the alkylacetylglycerol synthesized via the remodeling pathway, was also demonstrable. Unlike the species present in other tissues however, it was found to be sensitive to the presence of 10 mM DTT. The diacylglycerol CDP-choline cholinephosphotransferase species was also demonstrable and supported the synthesis of both PAF and phosphatidylcholine, in the absence of DTT, when only the synthesis of phosphatidylcholine was expected. It is hypothesized that the rabbit endometrium possesses active enzymes which may catalyze PAF synthesis via both the de novo and 'remodeling' pathways.