The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD (+) dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (Omega Km) or gentamycin (Omega Gm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3＇-O-phosphotransferase gene and the gentamycin-3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by Omega Km or nGm, and the functional vdh gene was replaced by vdh Omega Km or vdh Omega Gm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp, strain HRvdh Omega Km, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identity of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase.