Thiolutin reversibly inhibits growth and ribonucleic acid synthesis in Saccharomyces cerevisiae. It is now demonstrated that, at 5 mug/ml, thiolutin rapidly inhibits all incorporation of radioactive precursors into ribonucleic acid and protein in Escherichia coli, although the incorporation of deoxythymidine into deoxyribonucleic acid continues for some time. Concentrations of thiolutin of 5 mug/ml and above are bacteriostatic and do not lead to unbalanced growth, so that cell size remains constant. The antibiotic and its inhibitory effects are easily removed by washing, whereupon macromolecular synthesis and cell division resume unimpeded. These data are consistent with reversible inhibition of ribonucleic acid synthesis being the primary mode of action of thiolutin in E. coli, and suggest that thiolutin may be a useful tool for studies where such reversible inhibition is required.