The processing pathway for the MHC class II-restricted presentation of endogenous cytosolic Ag is distinct from the class I pathway since a cytosolic form of the influenza virus A hemagglutinin, expressed by a recombinant vaccinia virus, was presented by HLA-DR in a B cell mutant lacking the TAP1 subunit of the transporter for Ag presentation (TAP). In this report, two additional B cell mutants have been used to define the requirements of this TAP1-independent processing pathway. The first mutant, .61, lacks expression of both TAP1 and TAP2 genes, and of both LMP2 and LMP7 genes encoding proteasome subunits. As expected, class I-restricted presentation of the influenza virus matrix protein was totally deficient in mutant .61. In contrast, class II-restricted presentation of both the natural cytosolic matrix and the engineered cytosolic hemagglutinin proteins was functional in mutant .61. Thus, presentation of cytosolic Ag by class II molecules is independent of both TAP subunits and of the two MHC-encoded proteasome subunits. However, this endogenous processing pathway is dependent on at least one other function encoded in the class II region of the MHC as demonstrated with the second mutant, .174, in which a large deletion eliminates all expressed class II genes. Mutant .174 transfected with HLA-DR1 genes was previously shown to be defective in the presentation of exogenous Ag but normal in the presentation of short exogenous peptides. We show here that .174(DR1) is also defective in the presentation of cytosolic matrix and hemagglutinin proteins. This similar requirement for the class II-restricted presentation of either cytosolic Ag or internalized exogenous Ag suggests that both forms of Ag are ultimately targeted to the same cellular compartment for association with class II molecules.