ABSTRACT'. The objective of this study was to characterize the fatty acid biosynthetic pathway of the lactating human breast. Mixed cell populations, obtained by centrifugation of human milk, were enriched in breast epithelial cells by a selective adsorption procedure. Confirmation of the identity of the breast epithelial cells was obtained immunohistochemically. These viable breast epithelial cells incorporated radioactively labeled acetate predominantly into fatty acids with less than 16C atoms. The presence of the two key enzymes characteristic of the medium-chain fatty acid biosynthetic pathway of nonruminant~, fatty acid synthetase, and thioesterase 11, was demonstrated both qualitatively, by immunohistochemistry, and quantitatively, by enzyme assay. The results indicate that the lipogenic system of the human breast is qualitatively very similar to that of rats, mice, and rabbits, which also secrete milk fats containing medium-chain fatty acids. Quantitatively, however, the mammary fatty acid biosynthetic pathway appears to be less active in humans than in these other species. (Pediatr Res 19: 139-143, 1985) The presence of breast epithelial cells in human milk was first reported more than a quarter century ago (10). Although a number of investigators have been successful in culturing these cells (3,5,12,17,26,27,34), it appears that, to date, no serious attempt has been made to utilize such cells in studying metabolic and enzymological characteristics of the lactating human breast. We have recently attempted to use these cells in the study of the fatty acid biosynthetic pathway.Work in our laboratory with rodents has demonstrated that the mammary epithelium of these animals is uniquely able to synthesize the medium-chain fatty acids characteristic of milk fat (1, 2 1, 28). We and others (13-15, 19, 3 1, 32) have shown that the unique product specificity of the mammary gland lipogenic system in nonruminant mammals is attributable to the presence in the epithelium of a tissue-specific chain-terminating enzyme, medium-chain fatty acyl-S-4'-phosphopantetheine-fatty acid synthetase thioester hydrolase (trivial name, thioesterase 11).