This study was initiated to explore the quantitative and qualitative differences in milk total fatty acids and milk retinyl esters when either hydrogenated or nonhydrogenated fat is fed during pregnancy and lactation. Rats were fed diets containing 10% by weight of corn oil or partially hydrogenated corn oil. Milk was collected on d 1, 8 and 14 of lactation and analyzed for protein, total fatty acids, fatty acid pattern, and retinyl ester pattern. Whereas diet produced no quantitative differences in milk protein or total fatty acids, the pattern of milk fatty acids varied significantly. Rats fed corn oil produced milk having more medium-chain saturated fatty acids, less long-chain monoenoic fatty acids, and more polyunsaturated fatty acids compared to those fed hydrogenated corn oil. Rats fed hydrogenated corn oil produced milk fat having 21-26% of the trans fatty acid, elaidic acid. Significant differences were also observed with duration of lactation: medium-chain fatty acids increased three to fourfold between d 1 and 8, where cis-monoenes and polyunsaturated fatty acids declined. The pattern of milk retinyl esters strongly reflected, but was not identical to, that of total milk fat. Comparing d 14 milk from rats fed corn oil with that from rats fed hydrogenated corn oil, medium-chain esters of retinol constituted 24 and 11% of total retinyl esters, whereas saturated long-chain fatty acid esters constituted 52 and 44%, respectively. trans Fatty acid esters of retinol comprised 24% of vitamin A esters in milk of rats fed hydrogenated fat. These data provide evidence that the composition of milk retinyl esters, as well as that of total milk fat, is determined both by the type of fatty acids from diet and from diet-related differences in de novo synthesis of fatty acids within the mammary gland and other tissues.