Mouse B16 melanoma cells maintained in vitro in the presence of interferon (IFN)-α become resistant to the in vitro antiproliferative effects of IFN-α. However, IFN-α-treated mice inoculated with these in vitro IFN-treated cells (Bl6α^res cells) have significantly increased life spans (ILS) and significantly higher cure rates than IFN-α-treated mice inoculated with B16 cells. This unexpectedly greater sensitivity of B16α^res cells to the in vivo antitumor effects of IFN-α was evaluated by in vivo cell depletion experiments. Depletion of either activated peritoneal macrophages or cytotoxic T lymphocytes (CTL) reduced the ILS of IFN-treated B16α^res-inoculated mice to a level comparable to that of IFN-treated B16-inoculated mice. Depletion of natural killer (NK) cells did not affect the ILS for IFN-treated B16α^res-inoculated mice. These studies indicate that activated macrophage and CD8 cell function, but not NK cell function, is important for the enhanced antitumor effects induced by IFN-α against B16α^res cells. Macrophage killing was unlikely to be mediated by TNF-α or IL-1 as B16 and B16α^res cells were equally sensitive to TNF-α and insensitive to IL-1 in vitro. Further, H-2K antigen expression is significantly more readily inducible on B16α^res cells than on B16 cells, consistent with enhanced CD8-mediated killing due to increased MHC class I antigen expression.