Bacillus stearothermophilus reacted to pseudomonic acid-induced inhibition of isoleucine-transfer ribonucleic acid (RNA) acylation and to energy downshift caused by alpha-methylglucoside addition with accumulation of guanosine 3',5'-polyphosphates [(p)ppGpp] and restriction of RNA synthesis. In vitro studies indicated that (p)ppGpp was synthesized by two different enzymes. One enzyme, (p)ppGpp synthetase I, was present in the ribosomal fraction, required the addition of a ribosome-messenger RNA-transfer RNA complex for activation, and was inhibited by tetracycline and thiostrepton. It is suggested that (p)ppGpp synthetase I is comparable to the relA gene product from Escherichia coli and is responsible for (p)ppGpp accumulation during amino acid starvation. The other enzyme, (p)ppGpp synthetase II, was found in the high-speed supernatant fraction (S100). It functioned independently of ribosomes, transfer RNA, and messenger RNA and was not inhibited by the above-mentioned antibiotics. (p)ppGpp synthetase II is thought to be responsible for (p)ppGpp accumulation during carbon source downshift. The two enzymes differ in their Km values for adenosine triphosphate (ATP):2mM ATP for synthetase I and 0.05 mM ATP for synthetase II. They also have different molecular weights: apparent Mr of 86,000 (+/- 5,000) for synthetase I and 74,000 (+/- 5,000) for synthetase II.