In Yarrowia lipolytica incorporation of radioactive precursors into RNA or protein led to a similar increase both during the yeast mode of growth and during the yeast-mycelium transition, indicating that no changes in the rate of total synthesis of either macromolecule occur. Actinomycin D did not inhibit RNA synthesis and differentiation in this yeast. Neither cycloheximide nor puromycin inhibited protein synthesis in Y. lipolytica, whereas trichodermin did. Addition of trichodermin at time 0 of the yeast-mycelium transition inhibited mycelium formation, indicating that translation is necessary for germ tube formation. Inhibition of DNA synthesis by hydroxyurea prevented the yeast-mycelium transition. However, removal of hydroxyurea permitted reinitiation of DNA synthesis and differentiation, independently of the mode of growth. 5-Azacytidine inhibited yeast growth at a concentration of 50 μ M but had no effect on mycelium growth. Treatment of the purified DNA from the yeast and mycelial forms with HpaII or MspI led to an identical restriction pattern with remarkably discrete bands. Our results show that in Y. lipolytica, translation, transcription, and DNA replication are necessary for the morphological conversion.