Helper-free herpes simplex virus type-1 (HSV-1) amplicon vectors elicit robust immune responses to encoded proteins, including human immunodeficiency virus type-1 (HIV-1) antigens. To improve this vaccine delivery system, seven amplicon vectors were constructed, each encoding HIV-1 Gag under the control of a different promoter. Gag expression levels were analyzed in murine and human cell lines, as well as in biopsied tissue samples from injected mice; these data were then compared with Gag-specific T cell responses in BALB/c mice. The magnitude of the amplicon-induced immune response was found to correlate strongly with the level of Gag production both in vitro and in vivo. Interestingly, the best correlation of the strength of the amplicon-induced immune response was with antigen expression in cultured DC rather than expression at the tissue site of injection or in cultured cell lines. These findings may have implications for the generation of improved HSV-1 amplicon vectors for HIV-1 vaccine delivery.