The gene endA, encoding the periplasmic endonuclease I (EndoI) of Escherichia coli, was identified on a cloned chromosomal 1.5-kb HindIII fragment. The nucleotide sequence of the fragment revealed an open reading frame (ORF) coding for a polypeptide of 235 amino acids (aa). The ORF preceeded by a region with two possible promoter sites displays promoter activity when cloned into an expression vector. On the C-terminal side, two sequences with putative transcription termination function are present. The predicted aa sequence suggests the presence of a signal peptide of 22 aa and a signal peptide cleavage site. A cold-shock supernatant from cells harbouring a multicopy endA+ plasmid contained an approx. tenfold higher amount than wild-type cells of the DNA double-strand- and single-strand-cleavage activities characteristic of EndoI. The growth rate and viability of the cells was not affected. The predicted aa sequence of the ORF is 60 and 54% identical to the sequence of extracellular DNases from Vibrio cholerae and Aeromonas hydrophila, respectively.