The pit+ gene, encoding the phosphate (inorganic) transport system of Escherichia coli, was isolated from a library of E. coli genes inserted in the cosmid vector pHC79. A 25.5-kb chromosomal DNA fragment was shown also to carry the gor locus encoding glutathione oxidoreductase. Physical mapping placed the two genes about 10 kb apart, confirming bacteriophage P1 mapping of the 77-min region. Subcloning and deletion analysis indicated that the entire pit+ gene was located within a 2.2-kb Sal1-Ava1 fragment. The pit+ gene product was identified by SDS-polyacrylamide gel electrophoresis as a 39-kdal inner membrane protein by two methods: 35S-methionine-labelling of minicells carrying pit+ plasmids or plasmids from which all or part of the pit+ gene was deleted. Overproduction of the Pit protein using a thermoinducible "runaway" replication plasmid. Complementation of the pit-1 mutant allele using a unit-copy-number pit+ plasmid indicated that the pit-1 mutation is recessive. Strains carrying a multicopy pit+ plasmid show a 10-fold increase in the initial rate of phosphate uptake; however there is no change in the steady-state level of 32Pi accumulation.