The sulfur-containing antibiotic thiolutin has been shown to be a potent, reversible inhibitor of the growth of Saccharomyces cerevisiae. Viability was unaffected over the concentration range of 4 to 100 mug/ml. At concentrations as low as 2 mug/ml, the drug inhibited ribonucleic acid (RNA) and protein synthesis in whole cells and spheroplasts. At these low concentrations, protein synthesis continued for a short period of time after RNA synthesis was completely stopped. With higher drug concentrations (greater than 20 mug/ml) protein synthesis was inhibited; concentrations of thiolutin up to 100 mug/ml did not affect translocation or peptide bond formation in cell-free protein-synthesizing systems from yeast. The effect of thiolutin on the activity of partially purified deoxyribonucleic acid-dependent RNA polymerases was examined, and the drug was found to be a potent inhibitor of RNA synthesis in vitro. Inhibition was greatest when the polymerase was preincubated with thiolutin. Several mechanisms are discussed to explain the multiple effects of thiolutin on S. cerevisiae. Since the action of the drug is easily reversed, thiolutin may prove to be of use in studies of various stages of yeast growth.