The development of a serum-free, low-protein culture medium has allowed the detection of tumour-specific cytolytic cells in EMT6 immunized mice bearing EMT6 multicellular tumour spheroids. Spheroid associated (SAC) and peritoneal (PC) effector cells were specific to EMT6 as the target cell, not killing line 1, P815 or RIF-1. The natural killer (NK) cell sensitive target YAC-1 was also not lysed by SAC or PC, indicating undetectable levels of NK cells present within infiltrated spheroids. In contrast, high levels of cytolytic activity were present in SAC, PC and spleen cells against WEHI-164, a line sensitive to natural cytotoxic (NC) and macrophage mediated killing. The EMT6 specific activity was mediated by Thyl+, Lyt2+ cells. The anti-WEHI-164 effector cell population was Thyl-, Lyt2-. The WEHI-164 killer cells were present in SAC and PC from unimmunized mice while the EMT6 specific effector cells were not. After separation of SAC and PC by size using centrifugal elutriation, anti-EMT6 activity was present only in the lymphocyte fraction while anti-WEHI-164 activity was enriched in the macrophage fraction.