An extracellular ribonuclease of Trichoderma harzianum, named RNase Th, was purified to homogeneity by CM-cellulose chromatography followed by DEAE-cellulose and Sephadex G-50 chromatography. The enzyme consisted of one polypeptide chain of 102 residues. The molecular weight was found to be 11,000 by SDS/PAGE and 10,342 from the amino acid composition. The pH optimum was at 8.0 with yeast RNA and 6.0 with GpC. RNase Th had a unique high pI of 9.5. The enzyme degraded only poly(I), GpN, G > p, and I > p (2’,3’-cyclophosphate) substrates. Products were 3’-IMP (with poly(1)) and 3’-GMP (with GpN) via guanosine 2’,3’-cyclophosphate. It was concluded that RNase Th is a guanyI-specific cyclizing RNase (EC 3.1.27.3). The kinetic parameters of reactions with GpN were measured and shown to be similar to those of other fungal guanyl RNases.