The complete human T-cell leukemia virus type I (HTLV-I) env gene was inserted into an expression cassette containing the adenovirus 5 major late promoter (Ad5-MLP). Recombinant Ad5-HTLV-I-env was obtained by homologous recombination in 293 cells simultaneously transfected by the expression cassette and the genomic DNA of Ad5. In vitro expression of the HTLV-I-env gene in the recombinant vector was detected by immunofluorescence and Western blotting. Functional expression of HTLV-I-env was confirmed by syncitium formation specifically in HeLa cells infected with Ad5-HTLV-I-env. Two immunization regimens against HTLV-I were tested in WKY and Fischer F-344 rats. The first involved WKY rats primed with Ad5-HTLV-I-env or naked DNA plasmids containing the HTLV-I-env gene and boosted with Ad5 containing the HTLV-I-env gp46 gene or with baculovirus-derived recombinant gp46. No antibody against HTLV-I was detected, while HTLV-I-specific cytotoxic T lymphocytes were recovered from all immunized groups but not from controls. The second approach involved Fischer F-344 rats primed and boosted with recombinant vaccinia virus containing the HTLV-I-env gene. Such rats developed antibodies against the HTLV-I env gp21 and gp46 (non-neutralizing). After challenge with human HTLV-I-producing cells (MT-2), both immunization regimens were found to induce partial protection.