Mutations that cause the constitutive expression of the PHO regulon of Escherichia coli occur either in the pst operon or in the phoR gene, which encode, respectively, a high-affinity Pi transport system and a histidine kinase sensor protein. These mutations are normally selected on glycerol-2-phosphate (G2P) as the carbon source in the presence of excess Pi. The emergence of early PHO-constitutive mutants, which appear after growth for up to 48 h on selective medium, depends on the presence of phoA, which codes for a periplasmic alkaline phosphatase, while late mutants, which appear after 48 h, depend both on phoA and on the ugp operon, which encodes a glycerophosphodiester transport system. The emergence of the late mutants hints at an adaptive mutation process. PHO-constitutive phoR mutants appear only in a host that is mutated in pitA, which encodes an alternative Pi transport system that does not belong to the PHO regulon. The conserved Thr217 residue in the PhoR protein is essential for PHO repression.