The alpha-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, gamma-hexachlorocyclohexane (gamma-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of gamma-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS6100 composite transposon containing two copies of linB, and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other alpha-proteobacterial strains but not to any of the beta- or gamma-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for gamma-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in gamma-HCH degradation.