Chymotrypsin inactivation of lysophosphatidic acid acyltransferase activity in detergent-disrupted rat liver microsomes, but not in intact microsomes, falsely indicated a lumenal location for the enzyme. Inhibition by several other proteases in the absence of detergent showed that lysophosphatidic acid acyltransferase activity is located on the cytoplasmic surface of microsomes. Chymotrypsin inactivation did not occur in vesicles disrupted by nitrogen cavitation unless deoxycholate was present, suggesting that deoxycholate exposes a cryptic chymotrypsin cleavage site. Criteria for localization of lumenal microsomal enzymes should include studies using several proteases and/or employ more than one method of microsomal disruption.