Polyunsaturated fatty acids in human milk may derive from diet, liberation from maternal body stores, or endogenous synthesis from precursor fatty acids. The contribution of each of these sources has not been studied in detail. Although maternal diet is a key factor affecting human milk composition, other factors such as gestational age, stage of lactation, nutritional status, and genetic background are known to influence the fat content and fatty acid composition in human milk. Both linoleic and alpha-linolenic acids, the essential fatty acids, are present in human milk, as are several other n-6 and n-3 longer chain polyunsaturated fatty acids that are required for optimal growth and development of infants. The fatty acid profile of human milk from lactating women of different countries is remarkably stable, but there is variability in some of the components, such as docosahexaenoic acid, which is mainly due to differences in dietary habits. Tracer techniques with stable isotopes have been valuable in assessing the kinetics of fatty acid metabolism during lactation and in determining the origin of fatty acids in human milk. Based on these studies, the major part of polyunsaturated fatty acids in human milk seems not to be provided directly from the diet but from maternal tissue stores.