Genetic immunization using viral vectors provides an effective means to elicit antigen-specific cellular immune responses. Several viral vectors have proven efficacious in inducing immune responses after direct injection in vivo. Among them, recombinant, self-inactivating lentiviral vectors are very attractive delivery systems, as they are able to efficiently transduce into and express foreign genes in a wide variety of mammalian cells. A self-inactivating lentiviral vector was evaluated for the delivery of human immunodeficiency virus 1 (HIV-1) envelope sequences in mice in order to elicit specific immune responses. With this aim, BALB/c mice were immunized with a single injection of self-inactivating lentiviral vectors carrying either the full-length HIV-1(HXB2) Rev/Env (TY2-IIIBEnv) or the codon-optimized HIV-1(JR-FL) gp120 (TY2-JREnv) coding sequence. Both vectors were able to elicit specific cellular responses efficiently, as measured by gamma interferon ELISPOT and chromium-release assays, upon in vitro stimulation of splenocytes from BALB/c immunized mice. However, only the TY2-JREnv-immunized mice were able to elicit specific humoral responses, measured as anti-gp120 antibody production. These data provide the first evidence that a single, direct, in vivo administration of a lentiviral vector encoding a viral gene might represent a useful strategy for vaccine development.