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Human milk contains a lipase (bile salt-stimulated lipase) that is considered to have an important role in infant fat digestion. In this study we compared the characteristics of bile salt-stimulated lipase activity in milk samples from mothers delivering prematurely (26–30 and 31–37 weeks of gestation) and in milk from mothers delivering at term (38–42 weeks of gestation). Preterm milks were collected at day 1–5 and during week 6 of lactation. Term milks were collected during week 6 of lactation. The characteristics of the enzyme (kinetics. enzyme concentration, pH optimum, and pH stability, effects of bile salt structure and concentration, eserine inhibition) were identical regardless of length of pregnancy or duration of lactation. Bile salt-stimulated lipase had a neutral to alkaline pH optimum (pH 7.3–8.6), was stable for 1 h at a wide pH range (pH 3.1–8.6), was active only in the presence of primary bile salts, and was inhibited by eserine. The data indicate that, following parturition at as early as 26 weeks of gestation, the mammary gland synthesizes bile salt-stimulated lipase with identical characteristics as does the mammary gland after a full-term pregnancy.