We have determined the sequence of a DNA fragment containing the Escherichia coli serB gene. An open reading frame of 966 nucleotides was identified that encodes a polypeptide of 322 amino acids with a molecular weight of 35,002 daltons. The transcription start site was determined by Mung Bean nuclease mapping. The -10 and -35 regions of the serB promoter lack homology to the consensus sequences. In addition, the -35 region of the serB promoter overlaps the -35 region of a second divergent promoter. Frameshift mutations were constructed at three different sites within the serB gene. When plasmids carrying these mutations were used as templates in a minicell system, mutations closer to the proposed transcription and translation start sites resulted in smaller polypeptides than those further away, confirming the proposed direction of transcription and translation. The observed sizes of the truncated and native polypeptides were in agreement with those predicted from the DNA sequence. A very stable stem and loop structure (delta G= -32 kcal/mole) that does not fit the criteria of known transcription terminators was found one nucleotide downstream from the putative UAA translation stop codon.