Background. In clinical trials, canarypox ALVAC-human immunodeficiency virus (HIV) vaccines have been shown to elicit human HIV-specific cytotoxic T lymphocyte (CTL) responses in some but not all healthy uninfected adults. Methods. A clinical trial was conducted to examine whether the vaccine vCP1452 would elicit a greater HIV-specific CTL response when given at a dose of 10 8.0 TCID50 (60 participants) than when given at the regular dose, 107.26 TCID50 (40 participants); as a control, a placebo vaccine preparation also was administered (10 participants). Results. Two weeks after the last vaccination in a series, HIV-specific CTL responses were not significantly different when measured by either chromium-release assay (8% and 16% in the high- and regular-dose recipients, respectively) or interferon-γ ELISpot assay (8% and 15% in the high- and regular-dose recipients, respectively); moreover, recipients of the higher dose had greater local and systemic reactions (P < .001). Conclusions. High reactogenicity associated with an increased dose of vCP1452 negates the need for further evaluation of this strategy to boost the frequency of HIV-specific CTL response in seronegative human subjects. Development of highly immunogenic canarypox vectors requires further work to optimize vector and insert design, as well as novel ways to increase dosage and to reduce reactogenicity. © 2005 by the Infectious Diseases Society of America. All rights reserved.