Susceptibility testing by the broth dilution method showed that all the gram-positive but only some of the gram-negative bacteria tested were susceptible to the antitumor antibiotic, macromomycin (MCR; NSC 170105). The minimal inhibitory concentration for the susceptible organisms was less than 3 mug/ml. The action of MCR was bactericidal; however, at very high concentrations (50 mug/ml and above) some organisms occasionally escaped death. None of the escaped organisms was resistant to MCR. In combination with other commonly used antibiotics, MCR displayed partial synergy for Pseudomonas aeruginosa (from a minimal inhibitory concentration of >100 to 12.5 mug/ml with 100 mug of chloramphenicol per ml) and for Bacillus pumilus and Staphylococcus aureus (from 1.6 to 0.4 mug/ml and below) with polymyxin B. As with mammalian cells, (125)I-labeled MCR was irreversibly bound to both gram-positive and -negative bacteria. Treatment with trypsin of the (125)I-labeled MCR-exposed cells did not release the bound MCR or reverse its lethal effect. When in solution in a protective buffer at 4 degrees C, MCR was stable for up to 45 days; at 37 degrees C, however, 25% of its bactericidal activity was lost in 72 h. Loss of activity was enhanced 16-fold in the presence of both heated and unheated pooled human sera. Urine had no effect on the activity of MCR.