Recombinant vaccinia viruses containing either the entire gag/pol gene or the reverse transcriptase (RT) domain of the human immunodeficiency virus (HIV) were constructed. In mammalian cells infected with the recombinant vaccinia virus containing the gag/pol gene, major and minor polypeptides of 55 and 41 kDa were made, but processed gag products (p24/p17/p15) were not detected. In addition, none of the products of the pol open-reading frame were seen. Both the 55- and 41-kDa gag proteins were post-translationally modified by addition of myristic acid residues in recombinant vaccinia-infected cells, and were immunoprecipitated by antiserum to p24 gag, as well as by antisera from HIV-infected patients. These results indicate that neither proteolytic processing nor other HIV proteins are required for myristilation, and suggest that the 55- and 41-kDa gag precursors share the same amino terminus as p17. Cells infected with a separate vaccinia recombinant containing a truncated piece of the gag/pol gene with added start and stop codons at the 5' and 3' ends of the RT reading frame synthesized a major 61-kDa and a minor 51-kDa protein product which reacted immunologically with both a monoclonal antibody to native HIV p66/51 and antisera from HIV-infected patients. These proteins were purified from recombinant vaccinia-infected mammalian cells, and their enzyme activity was found to be similar to that of authentic HIV RT. Cells infected with the vaccinia/RT vector contained approximately 200-fold more RT per milligram of protein than cells infected with HIV. Recombinant RT was inhibited by dideoxynucleoside triphosphates and should be useful in screening for specific inhibitors of this enzyme. Mice inoculated intradermally with 10(8) plaque-forming units of the vaccinia/RT vector developed specific antibodies to the p66/51 proteins of HIV, but anti-HIV antibodies were not detected in mice inoculated with the vaccinia/gag vector.