The biochemical mechanisms and enzymes involved in the processing of protein antigens for presentation by major histocompatibility complex class II molecules are poorly understood. This work describes the purification of a cathepsin D-like enzyme isolated from the murine B lymphoma cell line A20, a model antigen presenting cell. Two forms of cathepsin D-like enzyme were detected. One is soluble and located in the lysosome-enriched sub-cellular fraction. The other is membrane-associated and located in the endosome-enriched fraction. The membrane-associated form was purified to apparent homogeneity by affinity chromatography on pepstatin A-Sepharose. Its apparent molecular weight is 48,000, and its pH optimum is pH 4.0. Endosomal cathepsins are known to be involved in antigen processing in vivo, and the purified membrane-associated cathepsin D-like enzyme from A20 cells was used to study antigen processing in vitro. The enzyme cleaved a model protein antigen, Staphylococcus aureus nuclease (Nase) and thereby generated antigenic fragments recognized by a Nase-specific T cell hybridoma. Such studies have allowed us to begin to understand the role of protease specificity and T cell determinant selection. © 1993 Academic Press, Inc.