A simple and rapid procedure for isolation of a highly purified ribonuclease from the culture filtrate of Trichoderma harzianum was described. The scheme included precipitation with ammonium sulfate followed by ion-exchange chromatography on DEAE-cellulose and CM-cellulose. The molecular weight of the RNase was found to be approximately 12,000 daltons by SDS-PAGE and gel filtration. The pH optimum for the enzyme was at 7.8 and maximum activity was obtained at 40–50°C