For extensive detection and monitoring of alkane-degrading bacteria, three combinations of PCR primer sets and gene probes were designed based on the homologous regions within a variety of alkane hydroxylase genes (alk genes) registered in the GenBank and examined their availability. PCR using the designed primers amplified DNA fragments with the expected sizes from all the tested bacterial strains used for primer design, and all of the amplified fragments gave positive results by Southern hybridization with the designed probes. The primers amplified DNA fragments with expected sizes from all the 76 wild-type newly isolated strains, while the probes gave positive results against the amplified fragments from 60 strains. Thus, the primer/probe system established in the present study can detect most of the alkane-degrading bacteria, and can be applied to evaluate alkane-degradation potential in the environments.Applying the designed primers, behavior of microbial populations responsible for the degradation of n-alkanes, a major component of crude oil, was monitored in seawater microcosm during the crude oil degradation process to clarify the degradation mechanisms of n-alkanes. It revealed that the alkane-degrading bacteria altered according to the fates of n-alkanes with different carbon chain lengths, so that shorter alkanes were degraded first by alkane-degrading bacteria possessing alk genes of Group I (Group I alkane-degrading bacteria) and longer ones afterwards by Group III alkane-degrading bacteria.