The foodborne pathogen Staphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes within S. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398-400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467-470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501-1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate of S. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.