It has been proposed that chitin synthase 3 (CHS3)-nediated chitin synthesis during the vegetative cell cycle is regulated by chitin synthase 4 (CHS4) of Saccharomyces cerevisiae. To investigate direct protein-protein interaction between the coding products of these two genes, a domain of Chs3p that is responsible for interaction with Chs4p was identified, using the yeast two-hybrid system. This domain of 54 amino acids, termed MIRC3-4 (Maximum Interacting Region of Chs3p with Chs4p), is well conserved among CHS3 homologs of various fungi. Some mutations in MIRC3-4 resulted in a decrease in the enzymatic activity and chitin contents. Chs3p carrying those mutations exhibited weak interactions with Chs4p, when assayed by the yeast two-hybrid system. Surprisingly, all the mutants were sensitive to Calcofluor regardless of changes in enzymatic activities or chitin contents. This report deals with a core region in MIRC3-4 that affects the interaction with Chs4p.