Staphylococcus aureus, the most salt-tolerant nonhalophilic bacterium, is the only foodborne pathogen that is able to grow at a levels below 0.90. The fundamental osmorgulatory strategy used by this organism involves the accumulation of intracellular compatible solutes such as proline or glycine betaine which are accumulated by transport and act as osmoregulators in cells. In this study, levels of proline transport systems and glycine betaine transport system of S. aureus were examined when cells are grown at high osmolarity. The levels of all three transport systems within S. aureus were elevated at high osmolarity and the most dramatic increase was found for the low-affinity proline transport system. However, in 5mM glycine betaine-supplemented medium, the level of the low-affinity proline transport system did not become elevated when cultures were grown at high osmolarity. The metabolic fate of the accumulated proline and glycine getaine was investigated by thin-layer chromatography an found to be not metabolized by S. aureus.