||A possible fate of duplicated genes is neofunctionalization, through which a new function is created that is different, although likely related, to the ancestral function. To test for neofunctionalization, a model system of three duplicate genes with unknown functions in the yeast species Candida glabrata was used. In Saccharomyces cerevisiae , a common model organism of yeast genomics, a well-studied signal transduction pathway (called the PHO pathway) regulates the transcription of a phosphate starvation inducible acid phosphatase, Pho5. In C. glabrata , a yeast species closely related to S. cerevisiae , there is also phosphate starvation inducible phosphatase activity regulated by a homologous PHO pathway; however, there is no PHO5 homolog in C. glabrata . Phosphatase activity during phosphate starvation is encoded in C. glabrata by the PMU2 gene, CAGL07546. PMU2 is very similar to both the C. glabrata PMU1 (CAGL07524) and PMU3 (CAGL07568) genes, and these genes are homologous to the PMU1 gene in S. cerevisiae . It is expected that these three genes are the result of a small-scale duplication event. When studying these similar duplicated genes, the question arises of how is the C. glabrata PMU2 gene unique? To answer this question, I sought to determine differences in the substrate specificity, function, and regulation of the genes. Instead of all three genes having similar phosphatase activity against organic phosphate substrates, I hypothesized that only Pmu2 has evolved to exhibit broad-range substrate specificity. Because PHO5 in S. cerevisiae is regulated by the transcription factor Pho4, I hypothesized that Pho4 also regulates only PMU2 . Finally, I hypothesized that because C. glabrata occupies a different niche than S. cerevisiae, C. glabrata has evolved different substrate specificity (relative to the hydrolysis of phosphate from organic phosphate containing compounds) that is better adapted to its' unique niche. By defining the differing characteristics of PMU1, PMU2, and PMU3, an evolutionary history was hypothesized and the role of neofunctionalization was ascertained. The data gathered suggests that because of selective pressures presented to C. glabrata in its' unique niche, Pmu2 has not evolved the ability to hydrolyze phytic acid, like S. cerevisiae Pho5. Instead, PMU2 has neofunctionalized to encode a phosphate starvation-inducible, broad-range, Pho4-regulated, acid phosphatase, whereas Pmu1 and Pmu3 are weaker, narrow-range phosphatases.