The basis for the difference between Campylobacter jejuni and Campylobacter coli is the presence and expression of the N-benzoylglycine amidohydrolase (hippuricase) gene only in C. jejuni. A pBR322 recombinant clone (pHIP-O) of C. jejuni TGH9011 capable of converting hippuric acid into benzoic acid and glycine, the hallmark of hippuricase activity, was characterized and sequenced. The hippuricase gene (hipO) was identified by use of deletion subclones and insertional inactivation. The transcription start point of the hippuricase gene was determined by primer extension analysis. A hippuricase-specific gene fragment was used to determine the presence of the gene in Campylobacter species. Maxicell analysis of recombinant plasmid pHIP-O by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the production of a 42-kDa protein corresponding to the HipO gene product, in excellent agreement with the predicted molecular mass of the protein.