Candida glycerinogenes WL2002-5 has a modest sugar tolerance and an extremely high glycerol productivity. Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, into the nuclear genome of a wide variety of host cells. In this study, we constructed the plasmid pZR and transferred it into A. tumefaciens LBA4404 to form the strain LBA4404-ZR. LBA4404-ZR was cocultivated with C. glycerologenesis, and putative transformants were identified by selection for zeocin resistance. Polymerase chain reaction and Southern blot analysis confirmed that the gene zeocin was integrated into the genome of engineered C. glycerologenesis. Optimization of the transformation condition was performed in darkness at 25 degrees C on induction medium for 24 h by cocultivation of C. glycerinogenes and LBA4404-ZR with a cell ratio of 1:500-1000. The transformation efficiency reached 2 transformants per 10(4) C. glycerologenesis cells. Our results demonstrated that A. tumefaciens-mediated transformation can be used for C. glycerinogenes. This transformation system can provide the basis for research of C. glycerologenesis in the future.