The catabolism of citronellol and geraniol [acyclic terpene utilization (Atu) pathway] was investigated in Pseudomonas citronellolis. A 13.3-kb genomic DNA fragment was cloned and harboured a putative regulator gene atuR and a gene cluster consisting of eight genes (atuABCDEFGH). Sequence analysis of the atu gene products showed a high degree of amino acid similarity (78-91% identity) to products of a similar gene cluster previously identified in Pseudomonas aeruginosa. Insertion mutagenesis in atuA resulted in inability of the bacteria to utilize acyclic terpenes as a sole source of carbon and energy and confirmed the involvement of atuA in the Atu pathway. Western blot analysis of wild-type and atuA mutant cells of P. citronellolis and P. aeruginosa for biotin-containing proteins enabled the identification of geranyl-CoA carboxylase (GCase), which is the key enzyme of the Atu pathway. GCase subunits were encoded by atuC and atuF. Putative functions for the other Atu proteins in the catabolic pathway of acyclic terpenes are discussed.