1. A mutant (ANT 8) of Schizosaccharomyces pombe which shows resistance to antimycin both in vivo and in vitro is characterized biochemically and genetically. 2. In crosses of ANT 8 with auxotrophic strains, resistance to antimycin segregates 2:2 indicating that resistance is conferred by a single nuclear gene. Diploids heterozygous for the resistance gene, however, show segregation of the resistance and sensitivity during mitosis. Possible reasons for this segregation are discussed. 3. Compared with the wild type, the NADH oxidase of ANT 8 requires 13 times as much antimycin for 95% inhibition. After addition of ubiquinone-3, electron transport which is less sensitive to antimycin is found only in the mutant. 4. The resistance of the mutant ANT 8 si due to the much weaker binding of antimycin to mitochondria. As in the wild type, two antimycin binding sites can be separated by binding studies. From the inhibition curves it is evident that binding of antimycin to oxidized mitochondrial particles does not correspond with its inhibitory effect on the partly reduced enzyme in kinetic studies. 5. The peak of the b-cytochrome absorbing at 560.2 nm at 77 degrees K in the wild type is shifted to 561 nm in the mutant. 6. A special preparation method for mutant mitochondrial particles is described, yielding highly active enzymes and CO-insensitive cytochromes. 7. The results are discussed with reference to the components in our model of the respiratory chain, which may be responsible for this type of resistance.