Rhodococcus opacus B-4 and B-9 are tolerant to various organic solvents including benzene, toluene, ethylbenzene, xylenes and styrene, and are suitable bacterial hosts for the production of chemical products from hydrophobic substrates. A 4.4-kb endogenous plasmid (pKNR01) was isolated from R. opacus B-4 and sequenced completely. Plasmid pKNR01 encodes proteins that share similarity to replication proteins from the enteric bacterial and actinomycete theta-replication plasmids. A 7.4-kb chimeric plasmid, designated pKNR01.1, was constructed by fusing Xhol-digested pKNR01 and Escherichia coli vector pSTV28. Plasmid pKNR01.1 had the ability to replicate in B-4 and B-9. A protocol for transformation of B-9 by electroporation was optimized employing pKNR01.1. Frequencies of 4.1 x 10(5) transformants per mu g of plasmid DNA were obtained for B-9 cells, whereas B-4 harboring naturally occurring pKNR01 was transformed at lower frequencies (approximately 1x10(4) transformants per mu g of plasmid DNA). Deletion analysis of pKNR01.1 showed that the 1.9-kb SphI-XhoI region containing the repA and repB genes and the 0.6-kb region upstream of repA was essential for plasmid maintenance in R. opacus strains.