A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons most frequently used in humans. The humanized pol gave dramatically improved levels of Rev-independent, in vitro protein production in mammalian cells and elicited much stronger cellular immunity in rodents than did virus-derived gene. Specifically, BALB/c mice were immunized with plasmids and/or recombinant vaccinia virus constructs expressing the synthetic gene. High frequencies of Pol-specific T lymphocytes were detected in these animals by the gamma interferon enzyme-linked immunospot assay against pools of short overlapping peptides. Characterization of the stimulatory peptides from these pools indicates that the optimized gene constructs are able to effectively activate both CD4+ and CD8+ T cells. Immunization of rhesus macaques with DNA vaccines expressing the humanized pol coupled to a human tissue plasminogen activator leader sequence led to pronounced in vitro cytotoxic T-lymphocyte killing activities and enhanced levels of circulating Pol-specific T cells, comparable to those observed in HIV-1-infected human subjects. Thus, optimizing the immunogenic properties of HIV-1 Pol at the level of the gene sequence validates it as an antigen and provides an important step toward the construction of a potent pol-based HIV-1 vaccine component.