Analysis of human milk was conducted to determine if free fatty acids occur naturally or as a consequence of artifactual lipolysis after milk expression. Five mothers provided triplicate early morning milk samples on day 43 of lactation. Following extraction, lipid classes were separated by preparative thin layer chromatography and quantified by capillary gas liquid chromatography. Fresh milk samples collected with 20 volumes chloroform-methanol (1:1, v/v) were analogous in total free fatty acid level and profile of fatty acids to a duplicate sample collected with 0.4M EDTA and immediately frozen at -10 C. Low milk levels of free fatty acids appear to exist naturally. During days 4-37 of lactation, four serial milk samples from 15 mothers were collected and frozen with 0.4M EDTA. The concentration of free fatty acids in colostrum (0.03-0.5%, w/w) was lower than for subsequent days (0.3-2.5%, w/w). Additional samples were collected with and without a lipase inhibitor (0.4M EDTA) and subjected to routine collection and storage procedures. Significantly different fatty acid profile and higher levels of free fatty acids in milk collected without a lipase inhibitor added indicate that domestic freezing and/or thawing ruptures the fat globule membrane, allowing sn-1-stereospecific serum stimulated lipoprotein lipase contact with its triglyceride substrate. Standard procedures for collection of human milk for gavage fed infants appears to stimulate artifactual lipolysis of milk triglyceride and subsequent release of free fatty acids. The proposed relationship between dietary free fatty acids and prolonged, unconjugated hyperbilirubinemia in the newborn is discussed with regard to the significance of preintestinal lipolysis.