Although intravesical instillation of bacillus Calmette-Guerin (BCG) is a clinically well-recognized therapy for bladder carcinoma in situ and recurrence prophylaxis, these mechanisms have not been fully understood. We studied the effects of BCG infection (Connaught strain) on target cancer cells and host immune systems in murine bladder cancer. The bladder cancer cell line, MB49, was used in C57/BL6 mice in vivo and in vitro. In vitro cytotoxicities against the cancer cell line were measured by 24-h 51Cr release assay. For effector cells, spleen mononuclear cells were obtained from mice injected intraperitoneally with BCG or BCG-infected irradiated MB49 cells. Although BCG infection of cancer cells did not affect the proliferation speed in vitro, the mice injected subcutaneously with BCG-infected MB49 cells survived significantly longer than those given untreated cancer cells. The mice surviving without tumor growth after injection of BCG-infected cancer cells could not reject a second injection of intact MB49 cells. In vitro cytotoxicity was enhanced by BCG infection of target cancer cells, but not by immunizing the mice with BCG from which effector cells were obtained. Moreover, cytotoxicity disappeared by depleting natural killer (NK) cells from effector cells. Although in vitro cytotoxicity was increased by immunizing the mice with BCG-infected irradiated MB49 cells, survival did not improve in these mice. These results suggest that a major part of BCG's anti-tumor effects can be attributed to the elimination of BCG-infected cancer cells by NK cells.