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The dimorphic fungus Yarrowia lipolytica grows to form hyphae either in rich media or in media with GlcNAc as a carbon source. A visual screening, called FIL (filamentation minus), for Y. lipolytica yeast growth mutants has been developed. The FIL screen was used to identify three Y. lipolytica genes that abolish hypha formation in all media assayed. Y. lipolytica HOY1, a gene whose deletion prevents the yeast-hypha transition both in liquid and solid media, was characterized. HOY1 is predicted to encode a 509-amino-acid protein with a homeodomain homologous to that found in the chicken Hox4.8 gene. Analysis of the protein predicts a nuclear location. These observations suggest that Hoy1p may function as a transcriptional regulatory protein. In disrupted strains, reintroduction of HOY1 restored the capacity for hypha formation. Northern blot hybridization revealed the HOY1 transcript to be approximately 1.6 kb. Expression of this gene was detected when Y. lipolytica grew as a budding yeast, but an increase in its expression was observed by 1 h after cells had been induced to form hyphae. The possible functions of HOY1 in hyphal growth and the uses of the FIL screen to identify morphogenetic regulatory genes from heterologous organisms are discussed.