In Saccharomyces cerevisiae, three chitin synthases have been detected. Chitin synthases I and II, the products of the CHS1 and CHS2 genes, respectively, are closely related proteins that require partial proteolysis for activity in vitro. In contrast, chitin synthase III is active in vitro without protease treatment, and three genes, CSD2 (= CAL1), CSD4 (= CAL2), and CAL3, are required for its activity. In the cell, the three enzymes have different functions. Chitin synthase I and II make only a small portion, < 10%, of the cellular chitin. In acidic media, chitin synthase I is required for normal budding. Chitin synthase II is required for normal morphology, septation, and cell separation. Chitin synthase III is required for the synthesis of 90% of the cellular chitin, including the chitin in the bud scars and lateral wall. Mutants defective in chitin synthase III are resistant to Calcofluor and Kluyveromyces lactis killer toxin, they lack alkali-insoluble glucan, and under certain circumstances, they are temperature-sensitive for growth. The available data suggest that many fungi have more than one chitin synthase and that these synthases are related to the S. cerevisiae CHS and CSD gene products.