The positive regulatory gene (phoM) for alkaline phosphatase of Escherichia coli was cloned on a mini-F plasmid pMF3 from the E. coli chromosome by a shotgun method. The hybrid plasmid pTHR32, which carries 10.8 kb chromosomal DNA, complemented both phoM and thrB mutations. The restriction map was constructed. Based upon this information, several PhoM- deletion plasmids and smaller PhoM+ plasmids were constructed in vitro. By examining the phenotypes and the physical maps of these plasmids, we could define the phoM gene locus in a 2.5 kb region on the restriction map of the cloned chromosomal DNA fragment. The PhoM+ plasmid not only enabled a phoM- -phoR- double mutant to express phoA (the structural gene for the alkaline phosphatase) but also phoB (another positive regulatory gene for phoA). These results are consistent with a model for genetic regulation of phoA expression that proposes that both the phoM and phoR gene products activate phoB expression under phosphate starved conditions, and PhoB protein, in turn, activates phoA expression. The phoM gene product was identified by the maxicell method as a protein with a molecular weight of 60,000. A hybrid plasmid that carries a phoM'-'lacZ fused gene on mini-F vector pMF3 was constructed in vitro. This plasmid enabled us to study phoM gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoM gene expression in these strains was studied under conditions of limited or excess phosphate. It was found that phoM expression was not regulated by phosphate nor by any of the pho genes. The transcriptional direction of phoM was found to be clockwise toward the thr operon on the E. coli genetic map. The fusion gene product interfered with phoB and phoA expression in the phoR mutants. Overproduction of PhoM protein increased phoB and phoA expression only in the phoR mutants. The implications of these findings are discussed.