A phase I randomized, double blind, placebo-controlled trial to assess the immunogenicity of a multiclade HIV-1 DNA plasmid vaccine was conducted in 31 HIV-1-negative Ugandans. Following immunization with DNA at 0, 1, and 2 months, the frequency of HIV-specific immune responses was assessed up to 10 months using a standard chromium release assay (CRA), lymphoproliferative assay (LPA), and antibody dependent cell-mediated cytotoxicity assay (ADCC). Seven of 15 (47%) vaccinees demonstrated CTL activity using the CRA to HIV-1 Env B with responses observed 1 month following the second vaccination and as late as 7 months following complete immunization. Additionally, lymphoproliferative reponses were observed in 14/15 vaccinees against p24. No CTL or LPA responses were observed at baseline or in the placebo group. ADCC activity was minimally induced by DNA vaccination. This study demonstrates that immunization with DNA alone induces CTL and lymphoproliferative responses in a population that will participate in a phase IIb study evaluating HIV-1 DNA priming followed by boosting with a replication-defective recombinant adenovirus vector.