We assessed the immunogenicities and efficacies of two highly attenuated vaccinia virus-derived NYVAC vaccine candidates encoding the human T-cell leukemia/lymphoma virus type 1 (HTLV-1) env gene or both the env and gag genes in prime-boost pilot regimens in combination with naked DNA expressing the HTLV-1 envelope. Three inoculations of NYVAC HTLV-1 env at 0, 1, and 3 months followed by a single inoculation of DNA env at 9 months protected against intravenous challenge with HTLV-1-infected cells in one of three immunized squirrel monkeys. Furthermore, humoral and cell-mediated immune responses against HTLV-1 Env could be detected in this protected animal. However, priming the animal with a single dose of env DNA, followed by immunization with the NYVAC HTLV-1 gag and env vaccine at 6, 7, and 8 months, protected all three animals against challenge with HTLV-1-infected cells. With this protocol, antibodies against HTLV-1 Env and cell-mediated responses against Env and Gag could also be detected in the protected animals. Although the relative superiority of a DNA prime-NYVAC boost regimen over addition of the Gag component as an immunogen cannot be assessed directly, our findings nevertheless show that an HTLV-1 vaccine approach is feasible and deserves further study.