Three new antibiotics isolated from broth cultures of a Pseudomonas were evaluated for antitumor activity against murine leukemias L1210 and P388. The antibiotic with the dichloromethyl group at the 3 position of actinobolin, an antibiotic produced by a Streptomyces, is the major product (Y-12278), and two analogs of Y-12278 are minor. When these antibiotics were administered i.p. on days 1 to 4 to mice bearing ascitic leukemias, the most effective was Y-12278, which increased the lifespan of mice implanted with leukemias L1210 and P388 by 88 and 110% over controls, respectively. On the same treatment schedule, less than 60% ILS (increase in lifespan) was obtained by oral and s.c. administration of Y-12278 to mice implanted i.p. with these leukemias, and by its i.p. injection to mice implanted i.c., i.v. and s.c. with leukemia L1210. With i.p. administration of Y-12278, a single injection on day 1 only was less effective in increasing the lifespan of mice bearing ascitic leukemias L1210 and P388 than the prolonged treatment schedules such as daily on days 1 to 4. Y-12278 administered i.p. on days 3 to 6 was shown to possess antitumor activity against i.p. implanted rat hepatomas. More than 200% ILS was seen in hepatomas AH44 and AH7974F.