A number of thiostrepton-resistant mutants of Bacillus subtilis were obtained. The thi mutations map proximally to strA. Effects of thiostrepton on polyphenylalanine synthesis with ribosomes and S-100 fractions from parent and mutant strains indicated that resistance was localized to the ribosomes. Furthermore, effects of thiostrepton on binding of [3H]GTP to ribosomes and 50S subunits from thiostrepton-sensitive and-resistant strains localized the site of resistance to the 50S subunit. In addition, revertants from thiostrepton-resistance to thiostrepton-sensitivity were obtained. Ribosomes and 50S subunits from these thiostrepton-sensitive revertants were sensitive to thiostrepton similar to parental sensitive B. subtilis.