Mutants unable to utilize acetate as sole source of carbon and energy were isolated from ethyl-methanesulphonate-mutagenized cultures of a prototrophic Bacillus stearothermophilus strain. Three groups of mutants had identifiable defects. Thirteen of the mutants lacked one or both enzymes of the inducible anaplerotic glyoxylate shunt, and three mutants were auxotrophic for isoleucine and valine when grown on acetate, in a manner analogous to acetohydroxyacid synthase I mutants of enteric bacteria. One mutant was defective specifically in acetate uptake, providing direct evidence for an acetate transport system in B. stearothermophilus. The acetate uptake system was uncoupler-sensitive and had a relatively high K(m) (in the range 300-350-mu-M). Escherichia coli was also shown to possess a saturable acetate uptake system, but its K(m) was much lower (15-mu-M).