Leukocytes contain an enzyme that detoxifies bacterial lipopolysaccharides (also called endotoxins) by removing fatty acyl chains that are attached in acyloxy acyl linkage to the glucosamine backbone of lipid A. We describe the purification of an enzyme that carries out this activity, termed acyloxyacyl hydrolysis, from the HL-60 human promyelocyte cell line. The enzyme is a glycoprotein of apparent Mr = 52,000-60,000 that is found in low abundance (less than 0.001% of the cell lysate protein), principally in the granule fraction of the cells. The protein has two disulfide-linked subunits of apparent Mr = 50,000 and 14,000-20,000, each of which contains N-linked oligosaccharides. This is the first lipopolysaccharide-degrading enzyme that has been purified from animal cells.