Mutants of Escherichia coli defective in the hexose phosphate transport system were isolated. Negative selection by penicillin treatment or positive selection with phosphonomycin was employed. These mutants grew normally on all carbon sources other than hexose phosphates. The map location of the mutations in 18 independently isolated mutant strains was investigated by transduction crosses. All of the mutations were found to lie in the same region of the chromosome, in the region represented by min 72 on the Taylor map. The order of the genes in this region was found to be mtl-cysE-pyrE-uhp-bgl-ilv. Revertants of some of the mutants exhibited altered regulatory control of this transport system.