The nucleotide (nt) sequences flanking the peptidyl-tRNA hydrolase-encoding gene (pth) of Escherichia coli were determined and analyzed. A coding open reading frame (ORF-3), identified just downstream from pth, had a deduced amino acid (aa) sequence homologous to a family of GTP-binding proteins. We found discrepancies between the ORF-3 sequence from a plasmid clone used in previous studies and another one derived from Kohara's phage collection. Two putative genes, ORF-4 and ORF-2, were also found upstream from pth. ORF-4 could code for a 393-aa peptide homologous to membrane-bound proteins. The nt sequence between ORF-2 and pth revealed the existence of a CAP-binding site correctly positioned to regulate the expression of ORF-2.