The glpD gene, which encodes aerobic sn-glycerol 3-phosphate dehydrogenase, and the glpR gene, which encodes a repressor that negatively regulates the expression of the g/p regulon, map near minute 75 on the linkage map of Escherichia coli K-12. In the present study, the nucleotide sequence of the 2895 base pair of DNA containing the glpD control region and the glpE, glpG, glpR genes was determined. The translation initiation codons with adjacent ribosome-binding sites were found for these four genes. The transcription start site of the glpD gene was identified 42 base pairs upstream from the proposed methionine start codon, preceded by a region containing typical -10 and -35 sequences found in bacterial promoters. A binding site for the cyclic AMP-cAMP receptor protein complex was located just upstream from the -35 sequence, centered at position -63. The interaction site for the glp repressor was identified by using DNase I footprinting. This region contained two tandemly repeated sequences which started at the -10 sequence and continued to position +38. The glp repressor contained 252 amino acid residues and had a molecular weight of 28,046 which was deduced from the nucleotide sequence. The position of the initiation codon was verified by determination of the amino acid sequence of the N-terminus of the purified gip repressor. The presumptive helix-turn-helix region of the repressor was located near the N-terminus (amino acids 22 to 41) at a poSition analogous to that found for the operator binding domain of other repressors such as the deo and Jac repressors. The recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system. The presumptive glpR recognition helix was changed to the deoR recognition helix and the sixth amino acid arginine of the recognition helix was changed to alanine by site-directed mutagenesis. The mutant forms of the repressor had a greatly reduced affinity for the glpD operators in vivo, determined by measuring β-galactosidase activity in a strain carrying a glpD-lacZ fusion.