The structural gene for the biosynthetic l-glycerol 3-phosphate dehydrogenase has been mapped at min 71.5 on the Escherichia coli chromosome. This gene (gpsA) is co-transduced with the xyl, mtl, and pyrE loci. Three-factor conjugational crosses and the transduction data indicate that the order of loci in this region of the chromosone is mtl, gltE, gpsA, gadR, gadS, pyrE. Study of a temperature-sensitive gpsA mutant possessing a dehydrogenase of increased thermolability indicated that gpsA is the structural gene for the dehydrogenase. All dehydrogenase-deficient strains tested were mapped very close to the gpsA locus. Attempts at genetic complementation analysis were unsuccessful.