The lysosome-associated membrane proteins (LAMP), found in the outer membrane of lysosomes and also in a multilaminar compartment that contains major histocompatibility complex class II (MHC II) proteins, are directed to their localization by a cytoplasmic carboxyl-terminal sequence. Our studies of the immune response to LAMP-targeted proteins has led to the application of a HIV-1 gp160/LAMP chimeric gene as a novel means to enhance the MHC II presentation of gp160. Immunofluorescence microscopy confirmed that the gp160/LAMP protein had a cellular localization corresponding to that of lysosomes. Pulse-chase analysis confirmed that the rates of synthesis of gp160/LAMP and wild type gp160 were comparable and that both proteins were processed to gp120 at similar rates. However, the gp160/LAMP was degraded more rapidly than the wild type gp160. MHC II-mediated T cell proliferation assays performed with cloned human cell lines showed that gp160/LAMP stimulated greater responses than did the wild type gp160. Moreover, mice vaccinated with recombinant vaccinia expressing gp160/LAMP had greater gp160-specific lymphoproliferation responses and higher titers of anti-V3 loop antibodies than mice vaccinated with recombinant vaccinia expressing wild type gp160.