A murine footpad tumor model was used to determine the cytotoxic activity, tumor specificity, phenotypic profile, and cytokine production of stimulated cells from draining lymph nodes (DLN). Popliteal DLN from 5-day-old P-815 footpad tumors were stimulated with 10(-7) M phorbol 12, 13-dibutyrate +5 x 10(-7) M ionomycin for 16 hr and cultured in IL-2 (20 units/ml) for 7 or 14 days without autologous tumor. Most cells in both groups were CD3+ (93% at Day 7, 99% at Day 14); however, the percentage of CD8+ cells increased as the cell population matured in the presence of low-dose IL-2. On Day 7, the phenotypic profile was 62% CD4+ and 29% CD8+, whereas on Day 14 it was 16% CD4+ and 81% CD8+. Similarly, in vitro cytokine production increased with time in culture. After 7 days, the level of tumor necrosis factor-alpha (TNF-alpha) was 220 pg/mL and the interferon-gamma (IF-gamma) production was 150 pg/ml. At Day 14 the TNF level had increased to 500 pg/ml, and IF production had increased to 350 pg/ml. These increases in the CD8+ population and in cytokine production correlated with the increase in the percentage of target cells killed by the DLN cells. Cytolytic activity against P-815 was only 13% on Day 7 but 39% on Day 14. Neither group of effector cells (Day 7 or Day 14) had any cytolytic activity against the syngeneic tumor cell line L-1210, demonstrating the tumor specificity of the DLN cells. We describe a model for generating tumor-specific cytotoxic T-cells that have significant cytokine production, which may account for previously described in vivo activity.