In Saccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by the CSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized as CSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomycete Neurospora crassa and have used a "reverse genetics" approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of a N. crassa gene(designated chs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those of S. cerevisiae and Candida albicans. N. crassa strains in which chs-4 had been inactivated by the Repeat-Induced point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in the chs-4RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme in N. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.