The glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12 was cloned into pACYC177 from a lambda glpD transducing phage. The recombinant plasmid, designated pSH55, carried a 7.4-kilobase-pair HindIII fragment containing the glpD and glpR genes. The glpD gene was subcloned into pACYC177 on a 4.4-kilobase-pair BamHI-HindIII fragment. Expression of the cloned glpD gene was regulated in the manner previously described for the chromosomal glpD gene. The position of glpD on this plasmid was determined by Tn1000 insertional inactivation experiments. The glpD gene product, a polypeptide of Mr 55,000, was detected in a maxicell system. Truncated polypeptides replaced the 55,000-molecular-weight polypeptide when plasmid derivatives harboring Tn1000 insertions that inactivate glpD were used as templates. The sizes of these polypeptides confirmed the previously determined direction of transcription and allowed estimation of the translation start site. Determination of the apparent Mr of a hybrid protein encoded by a glpD'-'lacZ fusion provided additional evidence for the position of the glpD control region. The amino-terminal 30 to 60 amino acids of this hybrid protein (provided by glpD) were sufficient for efficient membrane localization of glpD'-'lacZ-encoded beta-galactosidase activity. The glpD3 mutation was mapped within the glpD gene, providing additional evidence that glpD is the structural gene for aerobic sn-glycerol-3-phosphate dehydrogenase.