Intestinal acid lipase is an enzyme whose greatest specific activity is localized to the villus tips of the proximal intestine (Rao, R.H. and Mansbach, C.M. (1990) Biochim. Biophys. Acta 1043, 273-280). This suggests that it plays a role in the processing of dietary lipids. We purified the enzyme in order to better characterize it. Acid lipase was isolated from intestinal mucosa of rats by a combination of ammonium sulfate precipitation, butanol extraction and chromatography on DEAE Bio-Gel, CM Bio-Gel and Sephadex G-75. This resulted in a single protein of Mr 53,700 on SDS-polyacrylamide gel electrophoresis. The isolation scheme produced a 3344-fold purification resulting in an enzyme whose specific activity was 801 mumols/min per mg protein. The yield was 50%. The purified enzyme was stimulated (20-fold) by the addition of tauro- or glycocholate but no other conjugated bile acid. A sharp peak in activity occurred at pH 5.6. The pI of the enzyme was 6.2. The reaction products produced under prolonged incubation suggested that monoacylglycerol was not hydrolyzed since an overabundance of monoacylglycerol was found with respect to the amount of fatty acid produced. These results suggested that intestinal acid lipase is potentially important in the metabolism of dietary lipids. Its proportionate role awaits further documentation.