Aims: To investigate the alkane-hydroxylating system of isolate SP2B, closely related to Rhodococcus ruber DSM 43338T and uncharacterized so far for its alkane degradation genes. Methods and Results: Although isolate SP2B and reference strain can grow on by-products from hexane degradation, the type strain R. ruber was unable, unlike SP2B isolate, to use short-chain alkanes, as assessed by gas chromatography. Using PCR with specific or degenerated primers, inverse PCR and Southern blot, two alkane hydroxylase encoding genes (alkB) were detected in both bacteria, which is in agreement with their alkane range. The first AlkB was related to Rhodococcus AlkB7 enzymes and contains a nonbulky residue at a specific position, suggesting it might be involved in medium- and long-chain alkane oxidation. The second partial alkB gene potentially belongs to alkB5-type, which was found in bacteria unable to use hexane. Moreover, a partial P450 cytochrome alkane hydroxylase, thought to be responsible for the hexane degradation, was detected only in the isolated strain. Conclusions: Rhodococcus ruber SP2B should prove to be a promising candidate for bioremediation studies of contaminated sites because of its large degradation range of alkanes. Significance and Impact of the Study: This is the first thorough study on R. ruber alkane degradation systems.