Acyloxyacyl hydrolase, a leukocyte enzyme previously has been shown to catalyze the hydrolysis of secondary (acyloxyacyl-linked) fatty acyl chains from the nonreducing glucosamine of the lipid A region of rough Salmonella typhimurium lipopolysaccharide (LPS). We describe here the activity of this enzyme toward smooth S. typhimurium LPS and LPS from Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Acyloxyacyl hydrolase released the secondary acyl chains from all of these lipopolysaccharides, regardless of the location of the acyloxyacyl linkage on the diglucosamine backbone or the structure of the acyl chains. The two acyloxyacyl linkages present in each LPS molecule apparently were hydrolyzed separately, so that free fatty acids released from the different sites accumulated at different rates. The purified enzyme also removed greater than 90% of the secondary acyl chains in each LPS, indicating that the enzyme acts not only on intact LPS but also on LPS molecules that have only one secondary acyl chain. The enzyme did not release the glucosamine-linked 3-hydroxyacyl chains. The specificity and versatility of the enzyme for cleaving acyloxyacyl linkages suggest that it may be a useful reagent for studying the structure and bioactivities of lipopolysaccharides with diverse carbohydrate and lipid A structures.