Heat-killed Mycobacterium bovis strain Bacillus Calmette-Guérin cells were sonically disrupted, and their antitumor effects against the line 10 hepatocarcinoma in strain 2 guinea pigs were evaluated. When injected together with viable line 10 cells, there was complete suppression of tumor growth. Growth of tumor was also suppressed when line 10 cells were injected contralaterally at the same time as the vaccine mixture. Multiple intratumor injections of sufficient disrupted M. bovis strain B. Calmette-Guérin were therapeutically effective against 74% of 7-day-old tumors and against 40% of 14-day-old tumors. Surviving animals were usually resistant to subsequent rechallenges with line 10 cells but not to syngeneic L2C leukemia cells. By means of a competitive radioimmunoassay, antigenic determinants were detected that were expressed by disrupted but not by intact bacteria.