Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP alpha loop and alpha helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP(I206A), PhoP(R210A), PhoP(L209A), and PhoP(H208A)) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP(H205A) and PhoP(V204A)) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP(V202A) and PhoP(D203A), had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP(V190A), PhoP(W191A), PhoP(Y193A), PhoP(F195A), PhoP(G197A,) PhoP(T199A), and PhoP(R200A)) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP(W191A) and PhoP(R200A) could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short alpha helix 3, more similar to OmpR than to PhoB of Escherichia coli.