Staphylococcus aureus can grow virtually anywhere in the human body but needs to import proline through low- and high-affinity proline transporters to survive. This study examined the regulation of the S. aureus putP gene, which encodes a high-affinity proline permease. putP::lacZ and putP::lux transcriptional fusions were constructed and integrated into the genomes of several S. aureus strains. Enzyme activity was measured after growth in media with various osmolyte concentrations. As osmolarity rose, putP expression increased, with a plateau at 2 M for NaCl in strain LL3-1. Proline concentrations as low as 17.4 muM activated expression of the putP gene. The putP::lux fusion was also integrated into the genomes of S. aureus strains that were either SigB inactive (LL3-1, 8325-4, and SH1003) or SigB active (Newman and SH1000). SigB inactive strains showed increased putP gene expression as NaCl concentrations rose, whereas SigB active strains displayed a dramatic decrease in putP expression, suggesting that the alternative sigma factor B plays a negative role in putP regulation. Mice inoculated with S. aureus strains containing the putP::lux fusion exhibited up to a 715-fold increase in putP expression, although levels in the various murine organs differed. Moreover, urine from human patients infected with S. aureus showed elevated putP levels by use of a PCR procedure, whereas blood and some abscess material had no significant increase. Thus, putP is transcriptionally activated by a low-proline and high osmotic environment both in growth media and in murine or human clinical specimens.