In an assessment of antibiotic action on Staphylococcus aureus, we found that distinct changes in intracellular nucleotide pools occur depending on the antibiotic mode of action. In particular, we have quantitated the effect of antibiotics on pools of the nucleotide guanosine 3'-diphosphate, 5'-triphosphate (pppGpp). Intracellular pppGpp levels increased in response to treatment with the isoleucyl tRNA synthetase inhibitor mupirocin, the uncoupler carbonyl cyanide-m-chlorophenylhydrazone, and rifampicin. These compounds were distinguishable by the degree in which they increased the pppGpp pool and by their differential effect on the pools of other nucleotides. This technique has been used to confirm and to refute the expected mode of action of several compounds identified as possible inhibitors of tRNA synthetases. Our results provide the framework for using nucleotide analysis in the assessment of novel antimicrobial compounds with unknown modes of action.