Reintroducing URA3 to its native locus in Candida albicans not5, not3, bur2, and kel1 disruption mutants enabled us to directly compare strains with control strain CAI-12. We showed that URA3 position affected orotidine 5'-monophosphate decarboxylase activity, hyphal morphogenesis, adherence, and mortality in murine disseminated candidiasis. After URA3 was reintroduced to its native locus, only NOT5 could be conclusively ascribed a role in virulence.