A collection of hybrid plasmids carrying either the wild-type or mutated glpT gene was generated in vitro and used to characterize the glpT-dependent active transport system for sn-glycerol-3-phosphate in Escherichia coli K-12. Restriction endonuclease analysis and recloning of DNA fragments localized glpT to a 3-kilobase pair PstI-HpaI segment of DNA. Comparison of DNA carrying glpT-lacZ fusions with DNA carrying intact glpT allowed determination of the direction of transcription. Through characterization of the proteins synthesized by strains harboring hybrid plasmids carrying amber, missense, or deletion mutations in glpT, it was shown that glpT is a promoter-proximal gene in an operon consisting of at least two genes. The gene product of glpT, the sn-glycerol-3-phosphate permease, was found associated with the inner membrane. It could be solubilized by treatment with sodium dodecyl sulfate at 50 degrees C. Its molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was dependent upon sample treatment before electrophoresis. The apparent molecular weight was 44,000 when membrane fractions were heated to 50 degrees C; subsequent treatment at 95 degrees C modified the protein such that it migrated faster (apparent molecular weight = 33,000). Several missense mutations in glpT were negatively dominant over wild-type glpT, indicating that the active form of the permease is multimeric. A gene (named glpQ) promoter distal to glpT codes for a periplasmic protein. This protein had previously been named GLPT protein to indicate its relationship to the glpT gene. The present report demonstrates that it is not the gene product of glpT and is not required for active transport of sn-glycerol-3-phosphate.