The concentration of Mn2+ is 1,000-fold higher in secretions than it is at internal sites of the body, making it a potential signal by which bacteria can sense a shift from a mucosal environment to a more invasive site. PsaR, a metal-dependent regulator in Streptococcus pneumoniae, was found to negatively affect the transcription of psaBCA, pcpA, rrgA, rrgB, rrgC, srtBCD, and rlrA in the presence of Mn2+. psaBCA encode an ABC-type transporter for Mn2+. pcpA, rrgA, rrgB, and rrgC encode several outer surface proteins. srtBCD encode a cluster of sortase enzymes, and rlrA encodes a transcriptional regulator. Steady-state RNA levels are high under low Mn 2+ concentrations in the wild-type strain and are elevated under both high and low Mn2+ concentrations in a psaR mutant strain. RlrA is an activator of rrgA, rrgB, rrgC, and srtBCD (D. Hava and A. Camilli, Mol. Microbiol. 45:1389-1406,2002), suggesting that PsaR may indirectly control these genes through rlrA, while PsaR-dependent repression of psaBCA, pcpA, and rlrA transcription is direct. The impact of Mn2+-dependent regulation on virulence was further examined in mouse models of pneumonia and nasopharyngeal carriage. The abilities of ΔpsaR, pcpA, and ΔpsaR ΔpcpA mutant strains to colonize the lung were reduced compared to those of the wild type, confirming that both PcpA-mediated gene regulation and PsaR-mediated gene regulation are required for full virulence in the establishment of pneumonia. Neither PcpA nor PsaR was found to be required for colonization of the nasopharynx in a carriage model. This is the first demonstration of Mn 2+ acting as a signal for the expression of virulence factors within different host sites. Copyright © 2006, American Society for Microbiology. All Rights Reserved.