5a,6-Anhydrotetracycline was discovered to be unique among several tetracycline derivatives tested in its ability to inhibit RNA accumulation in vivo at low concentrations (20 μg/ml and less). In addition, in vivo protein, DNA, and guanosine 5′-diphosphate 3′-diphosphate (ppGpp) synthesis were completely inhibited by 20 μg/ml 5a,6-anhydrotetracycline. ppGpp decay in a spoT strain was inhibited by 20 μg/ml 5a,6-anhydrotetracycline but not at higher concentrations of the drug. The inhibition of RNA synthesis by 5a,6-anhydrotetracycline may be due, in part, to reduced UTP and CTP synthesis. The effects of tetracyclines on in vitro ppGpp synthesis by crude stringent factor in the absence of ribosomes were investigated. It was determined that, of six tetracyclines tested, four strongly inhibited the reaction (oxytetracycline, chlorotetracycline, dedimethylaminotetracycline, and tetracycline) whereas 5a,6-anhydrotetracycline gave a moderate inhibition and α-6-deoxyoxytetracycline resulted in only a slight reduction in ppGpp synthesis. It is proposed that tetracyclines interfere with factors involved in ppGpp metabolism and function.