A gene encoding phenylacetaldehyde dehydrogenase (PAD), the enzyme involved together with a copper-topaquinone-containing amine oxidase in the initial steps of 2-phenylethylamine catabolism, was located at 31.1 min on the Escherichia coli K-12 genetic map. It was immediately adjacent to the gene encoding the amine oxidase but transcribed in the opposite direction. The purified PAD acted almost equally well on phenylacetaldehyde, 4-hydroxyphenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde. It had a subunit size of 54 kDa and its deduced amino acid sequence was approximately 40% identical to various eukaryotic and prokaryotic aldehyde dehydrogenases. A third gene encoding a positive regulatory protein required for expression of the amine oxidase and PAD genes was located next to the PAD gene. A gene previously located in this position was reported to encode a second amine oxidase but this was not confirmed. The nucleotide sequence from 1447 to 1450 kb on the E. coli K-12 physical map has been determined.