We describe a method for deleting or modifying genes from the pathogenic fungus Candida glabrata as well as a companion vector for complementation or ectopic expression experiments. A linear deletion fragment generated by polymerase chain reaction was used to replace a gene of interest with the C. glabrata gene encoding imidazoleglycerol-phosphate dehydratase (HIS3). As test cases, the chromosomal loci of the C. glabrata genes encoding aminoimidazole ribonucleotide carboxylase (ADE2) and encoding isopropylmalate dehydrogenase (LEU2) were deleted. To facilitate application of the deletion technique to essential genes, we also constructed vectors to allow expression of a complementing copy of the wildtype gene under control of the copper-inducible C. glabrata metallothionein I (MT-1) promoter. One version of the vector carried the Saccharomyces cerevisiae centromere (CEN) and autonomously-replicating sequence (ARS) regions. The C. glabrata ADE2 and LEU2 genes and a transposon-derived neomycin/kanamycin resistance gene were successfully expressed from this vector, with expression of the ADE2 and LEU2 genes complementing the ADE2 and LEU2 deletion mutations, respectively. However, this vector showed regulated expression only for the ADE2 gene. A second version of the vector, which carried an additional C. glabrata CEN and ARS region for stable plasmid maintenance, did show regulated expression for the LEU2 and neomycin/kanamycin resistance genes. This deletion and expression system is potentially applicable to any C. glabrata gene and is amenable to high-throughput application. We anticipate that these tools will have broad utility in deletion or modification of specific C. glabrata genes. This approach is also applicable to other yeast fungi.