The Mycobacterium tuberculosis PhoP-PhoR two-component system is essential for virulence in animal models of tuberculosis. Genetic and biochemical studies indicate that PhoP regulates the expression of more than 110 genes in M. tuberculosis. The C-terminal effector domain of PhoP exhibits a winged helix-turn-helix motif with the molecular surfaces around the recognition helix (alpha 8) displaying strong positive electrostatic potential, suggesting its role in DNA binding and nucleotide sequence recognition. Here, the relative importance of interfacial alpha 8-DNA contacts has been tested through rational mutagenesis coupled with in vitro binding-affinity studies. Most PhoP mutants, each with a potential DNA contacting residue replaced with Ala, had significantly reduced DNA binding affinity. However, substitution of nonconserved Glu215 had a major effect on the specificity of recognition. Although lack of specificity does not necessarily correlate with gross change in the overall DNA binding properties of PhoP, structural superposition of the PhoP C-domain on the Escherichia coli PhoB C-domain-DNA complex suggests a base-specific interaction between Glu215 of PhoP and the ninth base of the DR1 repeat motif. Biochemical experiments corroborate these results, showing that DNA recognition specificity can be altered by as little as a single residue change of the protein or a single base change of the DNA. The results have implications for the mechanism of sequence-specific DNA binding by PhoP.