We have cloned CHS4, a gene that complements the resistance to Calcofluor of the Saccharomyces cerevisiae cal2 mutant. We show that CHS4 is allelic to the previously described SKT5 and CSD4 genes. CHS4 encodes a 696 amino acids protein with no potential transmembrane domain. chs4-null mutants are resistant to Calcofluor white and exhibit a considerable reduction in cell wall chitin and in chitin synthase III (CSIII) activity. Biochemical characterization of chitin synthase III from these null mutants indicates that the defect is due to a reduced V(max) of the enzyme. This defect can be overcome in vitro by trypsin treatment of the membrane preparations. Chs4p does not act as a transcriptional or translational regulator of CHS3, the gene coding for the catalytic subunit of CSIII activity, and we therefore propose that Chs4p would be an essential component of the CSIII complex, acting as a post-translational regulator of this activity. In addition to the chitin defect, the chs4 mutant shows a severe defect in mating.