Bovine pancreatic lipase was isolated in pure form by lyophilization of fresh bovine pancreas, extraction of the enzyme with sucrose solution, fractional precipitation with ammonium sulfate and acetone, followed by chromatography on Sephadex G-100. The specific activity of the purest lipase fraction was 1750 micromoles fatty acid, liberated in 30 min per milligram of protein, indicating a purification of approximately 473-fold, with an overall yield of about 42%. Homogeneity of the enzyme was confirmed by rechromatography on Sephadex G-100 as well as with the gel electrophoretic and ultracentrifugal techniques. The purified enzyme gave a typical protein ultraviolet absorption spectrum with maximum absorption at 276 nm and minimum at 252 nm. The purified enzyme exhibited a single pH optimum of 8.8 and an isoelectric point near pH 5.5. Its optimum temperature was 37 C, and its optimum substrate concentration was 10%. These properties resembled those of milk lipase.