As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S. cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively. In S. cerevisiae, ScCHS1, ScCHS2, and ScCHS3 are all nonessential genes; only the simultaneous disruption of ScCHS2 and ScCHS3 is lethal. The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C. albicans. To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p. RO-09-3143 inhibited CaChs1p with a K i value of 0.55 nM in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine. RO-09-3143 also hampered the growth of the C. albicans cells with an MIC 50 value of 0.27 M. In the presence of RO-09-3143, the C. albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C. albicans Chs1p in septum formation. Although the effect of RO-09-3143 on the wild-type C. albicans was fungistatic, it caused cell death in the cachs2⌬ null mutants but not in the cachs3⌬ null mutants. Thus, it appears that in C. albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.Candida albicans is an opportunistic pathogen and is one of the most common pathogens in humans. In healthy individuals, it remains in the oral cavity, gastrointestinal tract, and genitalia; however, it colonizes and invades various tissues and organs and causes systemic fungal infections in neutropenic individuals, such as AIDS patients and those undergoing cancer chemotherapy or immunomodulation therapy for organ transplantation. Polyenes and azoles are used to treat systemic Candida infections, but the adverse effects of polyenes and the emergence of Candida strains resistant to azole compounds make the treatment of patients with systemic mycosis difficult (1).Chitin is one of the essential components of the fungal cell wall. As in Saccharomyces cerevisiae, C. albicans harbors three chitin synthases, which are encoded by distinct genes called CaCHS1 (2), CaCHS2 (3), and CaCHS3 (4, 5). According to the sequences and functional analogies, CaChs1p, CaChs2p, and CaChs3p are considered to correspond to S. cerevisiae Chs2p (ScChs2p), Chs1p (ScChs1p), and Chs3p (ScChs3p), respectively (2-13). CaChs2p is the most abundant protein among the three C. albicans chitin synthases (13), but it is not essential for viability, hyphal growth, or virulence (12, 14). CaChs1p and CaChs3p are required for septum formation and for a large part of the cell wall synthesis, respectively (11, 14). The expression of CaCHS2 and CaCHS3 is up-regulated during the morphogenetic transition from yeast to hyphae (3, 4, 13), whereas the expression of CaCHS1 remains low in both the yeast and hyphal forms (3). Although the cachs3⌬ null mutants retained the ability ...