Recently, APCs expressing genes for the alpha- and beta-chains of MHC class II, but not other proteins encoded in the MHC class II locus, have been found to be defective in processing proteins by the MHC class II Ag presentation pathway. Ag presentation of hen egg OVA has been examined in one of these cell lines, T2.Ak. OVA was processed normally by T2.Ak when compared with OVA processed by T1.Ak, a cell line that expresses the MHC genes missing in T2.Ak. By contrast, native hen egg lysozyme (HEL) was not presented as Ag by T2.Ak, which is in agreement with earlier results. Digestion with thiol proteases is important for Ag processing of HEL. We therefore analyzed the expression of these enzymes in T1.Ak and T2.Ak by using a reagent that specifically radiolabels thiol proteases. In these experiments, the repertoire of proteases expressed in the microsomes of T1.Ak was found to be distinct from the repertoire expressed by T2.Ak. Finally, in in vitro digestion experiments, the group of thiol proteases active in T1.Ak microsomes digested HEL differently from the group identified in T2.Ak. These results provide evidence that the defect in Ag presentation that is encoded by MHC genes and manifests itself in defective processing of HEL is not absolute. Further, the mutation in T2.Ak coincides with altered activities of thiol proteases, a class of enzymes involved in processing exogenous Ags.