Frozen storage is often used by milk banks to preserve expressed human milk for later use. Optimal storage and handling conditions which ensure minimum alteration of lipid composition have not been well defined. Therefore we investigated the effect of rapid freeze−thawing and storage conditions (−20 and −70° C) on the free fatty acid (FFA) levels and on the activities of lipoprotein lipase (LPL) and bile salt−stimulated lipase (BSSL) in human milk. Since during mechanical expression leakage of serum components into milk may occur, we also investigated the effect of the presence of serum on human milk LPL during storage. Lipase activity levels were unaffected by rapid freeze−thawing (x3) followed by storage for 1 month at −20 or −70° C. LPL activity (nmol FFA released/ml milk/min) was 414 ± 128, 451 ± 37, and 351 ± 20 and BSSL activity (μmol FFA/ml milk/min) was 5.7 ± 0.7, 5.5 ± 0.8, and 5.7 ± 0.2 in fresh, freeze−thawed, and stored milk, respectively. FFA levels (% of total lipid) were 3.01 ± 1.05 and 10.3 ± 1.6 in fresh−frozen milk stored at −7 0 and −20° C for 5 months, and 3.78 ± 1.08 and 13.60 ± 1.25 in specimens of freeze−thawed (x3) before storage at −70 or −20° C. Addition of serum had no effect on milk LPL at either temperature. We conclude that LPL and BSSL remain fully active during frozen storage of human milk and that milk fat is hydrolyzed at −20° C but not at −70° C. We suggest that banked human milk be stored routinely at −70° C.