A gene bank of Sau3A partially digested Candida albicans DNA in vector YEp13 was used to complement a ura3 mutation (orotidine-5′-phosphate decarboxylase, OMPdecase) in S. cerevisiae. Two plasmids which complemented ura3 and showed clear linkage of Ura⁺ and plasmid markers were selected for further study. Both plasmids also complemented the corresponding OMPdecase mutation (pyrF) in E. coli. Restriction mapping and subcloning studies localized the OMPdecase complementing activity to a region common to both plasmids. Probes prepared from this common region hybridized specifically to C. albicans DNA and not to E. coli or S. cerevisiae DNA. Southern blot analysis also showed that the restriction map of the ura3 complementing region of one plasmid was colinear with C. albicans genomic DNA. Expression of the OMPdecase complementing gene in E. coli and S. cerevisiae was not dependent upon orientation relative to vector sequences, suggesting that promotion could be occurring within the C. albicans fragment. Expression was sufficient to allow complementation in S. cerevisiae with integrating as well as high copy number vectors.