A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized. Partial amino-acid sequences from the purified 33-kDa chitinase were obtained. The amino-terminal peptide sequence was employed to design an oligonucleotide probe and was used as a primer to isolate a 1.2-kb cDNA. The cDNA codes for a protein of 321 amino acids, which includes a putative signal peptide of 19 amino acids. All microsequenced peptides found in this sequence, indicate that this cDNA codes for the 33-kDa chitinase. A high homology (approximately 43% identity) was found with fungal and plant chitinases, including yeast chitinases. However enzyme characteristics suggest a nutritional (saprophytic or mycoparasitic), rather than a morphogenetic, role for this chitinase. The chit33 gene appears as a single copy in the T. harzianum genome, is strongly suppressed by glucose, and de-repressed under starvation conditions as well as in the presence of autoclaved mycelia and/or fungal cell walls. The 33-kDa chitinase seems to be very stable except under starvation conditions. The independent regulation of each of the chitinases in T. harzianum indicates different specific roles.