ABSTRACTIt has been proposed that the ancestral fungus was mating competent and homothallic. However, many mating-competent fungi were initially classified as asexual because their mating capacity was hidden behind layers of regulation. For efficientin vitromating, the essentially obligate diploid ascomycete pathogenCandida albicanshas to change its mating type locus from heterozygousMTLa/α to homozygousMTLa/aorMTLα/α and then undergo an environmentally controlled epigenetic switch to the mating-competent opaque form. These requirements greatly reduce the potential forC. albicansmating. Deletion of the Yci1 domain geneOFR1bypasses the need forC. albicanscells to change the mating type locus from heterozygous to homozygous prior to switching to the opaque form and mating and allows homothallic mating ofMTLheterozygous strains. This bypass is carbon source dependent and does not occur when cells are grown on glucose. Transcriptional profiling ofofr1mutant cells shows that in addition to regulating cell type and mating circuitry, Ofr1 is needed for proper regulation of histone and chitin biosynthesis gene expression. It appears thatOFR1is a key regulator inC. albicansand functions in part to maintain the cryptic mating phenotype of the pathogen.IMPORTANCECandida albicansis a human fungal pathogen with a recently discovered, highly cryptic mating ability. For efficient mating, it has to lose heterozygosity at its mating type locus. Then,MTLhomozygous strains can undergo an epigenetic switch to an elongated yeast state, termed the opaque form, and become mating competent. This infrequent two-step process greatly reduces the potential for mating; few strains areMTLhomozygous, and the opaque state is unstable at the temperature of the mammalian host.C. albicanshas a complex mechanism for mating that appears designed to ensure that mating is infrequent. Here, we have characterized a new gene, opaque-formation regulator 1 (OFR1). Deleting theOFR1gene allowsMTLa/α strains to mate efficiently with either mating type or even mate homothallically. It is possible that downregulatingOFR1in the host environment could allow mating inC. albicansby a route that does not involveMTLhomozygosis.