Our previous reports established that immunization of mice in the footpad with a 15-amino acid synthetic peptide (R15K) from the V3 loop region in the envelope protein gpl20 of human immunodeficiency virus type 1 (HIV-1) resulted in rapid induction of major histocompatability complex (MHC) class I-restricted, CD8⁺ HIV-1 envelope-specific cytotoxic T lymphocytes (CTLs) in the proximal popliteal lymph node. While efficient CTL activity could be assayed in lymph node cells for 8 to 10 weeks after a single injection, spleen cells from these mice showed low to negligible levels of specific CTLs at 4 to 8 weeks postimmunization. We tested immunizing mice with a noncovalent mixture of a helper T cell (Th) activity-inducing peptide and R15K and observed efficient induction of R15K-specific CTL response that could be assayed up to 8 weeks postimmunization in cells obtained from both lymph node and spleen. Efficient CTL priming was observed when Th peptides from either of two different conserved regions in the HIV env were mixed with R15K, containing a dipalmityl–lysine–glycine–glycine moiety at the amino terminus. These data confirm reports in literature describing requirement of Th activity for efficient priming of CTL response in vivo. Additionally, these studies strongly suggest the possibility of formulating potential vaccine candidates consisting of mixtures of synthetic peptides capable of inducing Th and CTL responses in the context of multiple MHC haplotypes.