The well-characterized P815 tumor model was used to optimize anti-tumor immunization approaches in mice. Tumor peptides derived from antigens P198 or P1A were targeted to antigen-presenting cells (APC) by ex vivo pulsing. Initial experiments with irradiated pulsed splenic dendritic cells (sDC) injected weekly in the hind footpads for 3 weeks demonstrated cytolytic T lymphocyte (CTL) generation in 10-20% of mice. Because of the importance of interleukin-12 (IL-12) in tumor rejection responses, pulsed sDCs also were given together with recombinant murine IL-12 (rmIL-12). This strategy induced peptide-specific CTL in 100% of the mice. The IL-12 had to be injected in the footpads on days 0, 1 and 2 of each immunization week to achieve an optimal effect. The improvement seen with the addition of IL-12 prompted examination of other sources of APC. Purified resting B cells, lipopolysaccharide (LPS) blasts and nonfractionated splenocytes or peripheral blood mononuclear cells (PBMC) were pulsed with peptide and administered with the same schedule of rmIL-12. Because these cell types appeared to bind peptides less avidly than did DC, increasing peptide doses were used during pulsing. Interestingly, immunization with each of these APC also induced specific CTL in 100% of mice, provided rmIL-12 was coadministered. CTLs were detected both in the spleen and in the peripheral blood. Immunization with irradiated, P1A-pulsed PBMC plus rmIL-12 resulted in protection against challenge with tumors expressing the specific antigen in all mice. The ease by which human patient PBMCs can be prepared provides a straightforward vaccination approach to be used in clinical trials of peptide-based immunization in melanoma.