Streptococcus sanguinis is one of the most common agents of infective endocarditis. Spx proteins are a group of global regulators that negatively or positively control global transcription initiation. In this study, we characterized the spxA1 gene in S. sanguinis SK36. The spxA1 null mutant displayed opaque colony morphology, reduced hydrogen peroxide (H(2)O(2)) production, and reduced antagonistic activity against Streptococcus mutans UA159 relative to the wild type strain. The ΔspxA1 mutant also demonstrated decreased tolerance to high temperature, acidic and oxidative stresses. Further analysis revealed that ΔspxA1 also exhibited a ∼5-fold reduction in competitiveness in an animal model of endocarditis. Microarray studies indicated that expression of several oxidative stress genes was downregulated in the ΔspxA1 mutant. The expression of spxB and nox was significantly decreased in the ΔspxA1 mutant compared with the wild type. These results indicate that spxA1 plays a major role in H(2)O(2) production, stress tolerance and endocarditis virulence in S. sanguinis SK36. The second spx gene, spxA2, was also found in S. sanguinis SK36. The spxA2 null mutant was found to be defective for growth under normal conditions and showed sensitivity to high temperature, acidic and oxidative stresses.