A gene cluster involved in the utilization of tetrahydrofuran by Pseudonocardia sp. strain K1 was cloned and sequenced. Analysis of a 9.2-kb DNA fragment revealed eight ORFs. The genes designated as thmADBC encode the components of a putative monooxygenase exhibiting a high similarity to different binuclear-iron-containing multicomponent monooxygenases. thmA encodes the derived 545-amino-acid oxygenase alpha-subunit, thmD the 360-amino-acid reductase component, thmB the 346-amino-acid oxygenase beta-subunit, and thmC the 117-amino-acid coupling protein. Upstream of the thm genes, an additional ORF ( sad) was identified coding for a protein with high similarity to various aldehyde dehydrogenases. A succinate semialdehyde dehydrogenase activity was specifically expressed in tetrahydrofuran-grown cells. N-terminal sequence analysis of the purified protein revealed that it is encoded by sad. Northern blot analysis indicated that transcription of the thm genes and sad was specifically induced during growth on tetrahydrofuran. Mono-, di- and polycistronic transcripts of these genes were detected. Primer-extension analysis identified transcriptional start sites 37, 61, and 41 bp upstream of the translation start of sad, thmA, and thmB, respectively. Additional ORFs were identified upstream ( orfY) and downstream ( orfZ and aldH) of the thm genes. Furthermore, the data indicated that the analyzed gene cluster was present as a single copy and located on a plasmid.