The present study was undertaken to examine the immunogenicity of a single plasmid DNA representing the reverse transcriptase (RT) of HIV-1. Plasmids containing the enzymatically active RT as well as a mutated nonenzymatically active RT with nucleotide (nt)-binding motifs of YMDD and YMLL, respectively, were used to immunize mice. Both constructs induced similar good antibody and T cell responses, with a tendency towards antibody directed to peptides representing the active and mutated sites. Immunized mice were challenged with a murine pseudotype HIV-1/MuLV infected spleen cells. Seven out of 10 mice immunized with RT had no recoverable HIV-1, while 10 individuals immunized with the RT mutant and all the 18 controls had high levels of recoverable HIV-1. This indicates that mutation of RT reduces the desired immunogenicity.