This study is the first to analyse the genome of A. butzleri NCTC12481. By using one- and two-dimensional Pulsed Field Gel Electrophoresis in combination with Southern hybridisation the A. butzleri genome size was estimated (2.5 Mb) and a physical map was constructed, containing 35 restriction enzyme recognition sequences and 14 gene markers (5 copies of the genes for 16S rRNA and 23S rRNA’ 2 copies for glyA; one for rpoC and rpoB). In the attempt to identify virulence factors of A. butzleri, a homologue of pglF, a gene involved in N-linked protein glycosylation in C. jejuni, was found. Although the genes surrounding pglF in A. butzleri did not reveal a cluster order as in C. jejuni, the intriguing possibility for protein glycosylation in A. butzleri (perhaps involving different mechanisms) remains, since a glycosylated protein was detected by Alcian blue staining of an outer membrane protein extract. Three major proteins of the A. butzleri outer membrane extract were identified as PorA, FlaB and CadF which are known to play a role in pathogenicity in other bacteria. Electron microscopic analysis revealed a polysaccharide capsule on the surface of A. butzleri. In vitro studies of the interaction of A. butzleri with epithelial cultured cells (Caco-2) demonstrated the ability of A. butzleri to attach to and to invade Caco-2 cells. In order to develop a method for transposon mutagenesis of A. butzleri, a Himar1 transposon delivering vector carrying C. jejuni kanamycin resistance gene (aphA type III), was constructed. Attempts to introduce the vector into A. butzleri by electroporation, conjugation or heat shock transformation all failed. The obtained results provide an important foundation for further investigation of this organism and contribute towards broadening the knowledge of this potential foodborne pathogen.