UK-2A, a novel antifungal antibiotic, is a structural relative of antimycin A₃ (AA) and its mode of action is similar to that of AA which inhibits mitochondrial electron transport at complex III. In spite of their structural resemblance, AA had strong cytotoxicity while UK-2A had little cytotoxicity against LLC-PK1 cells as well as other types of cultured cells. When cells were treated with UK-2A or with A A the intracellular ATP content decreased significantly within 5 minutes in glucose-free medium to almost the same extent in both cases. Moreover, under the same conditions, UK-2A killed cells at a similar rate to AA. This suggested that UK-2A entered into the cells and, like AA, inhibited mitochondrial electron transport. On the other hand, AA stimulated reactive oxygen species (ROS) production within 5 minutes even at a low concentration of 1 μM whereas UK-2A did not show such an effect. The difference in the ROS-producing abilities of UK-2A and AA may account for the different cytotoxic effects of the two compounds.