A mutation in the phoP/phoQ locus (pho-24) that results in unregulated expression of PhoP-activated genes (phenotype PhoP constitutive [PhoP(c)]) was mapped to phoQ. Change of a Thr to Ile at position 48 of PhoQ was responsible for the PhoP(c) phenotype (attenuation of mouse virulence, defects in epithelial cell invasion, and macrophage spacious phagosome formation). PhoP phosphorylation by membrane extracts required PhoQ, and PhoQ Ile-48-containing membranes demonstrated increased net phosphorylation of PhoP.