The nucleotide sequence of the cloned DNA, 1,243 bp in length coding for N-acetylneuraminate lyase (N-acetylneuraminate pyruvate lyase; NPL) of Escherichia coli has been determined. Nucleotide sequence and amino acid analysis have assigned the open reading frame for NPL, starting with the ATG near its 5'terminus. The molecular weight calculated from the predicted amino acid sequence was 32,640 daltons, being in good agreement with that of a NPL subunit estimated by the SDS-PAGE method and amino acid composition. Several signal sequences conserved in the promoter regions of E. coli were found in the npl gene. They were the Shine-Dalgarno sequence, the Pribnow box and the sequence coserved in the "-35 region" and they were separated to each other with preferable spacing for an efficient transcription. Downstream from the termination codon, the inverted repeat sequence was present, followed by 4 successive T's.