After nitrosoguanidine mutagenesis we isolated from mouse leukemia L5178Y cells a mutant cell (Bla-R) resistant to blasticidin S, an inhibitor of protein synthesis. Neither growth nor leucine incorporation into hot-acid insoluble fraction of Bla-R cell was inhibited by 5 to 20 microgram/ml blasticidin S, which almost completely blocked protein synthesis as well as growth of the parental L5178Y cells. However, other inhibitors such as fusidic acid, cycloheximide, ricin D or L-asparaginase blocked protein synthesis in Bla-R cells to the same extent as in L5178Y cells. Protein synthesis in vitro using S-30 extracts from the parental cell line L5178Y was almost completely blocked in the presence of the antibiotic, while no inhibition by blasticidin S occurred when S-30 extracts -rom Bla-R mutant cells were used. Protein synthesis assays were made by using the S100 fraction from rat liver together with ribosomes from either L5178Y cells or Bla-R cells. Blasticidin S inhibited protein synthesis when ribosomes were derived from L5178Y cells, but not from Bla-R mutant.