Tyrostatin, a new carboxyl proteinase inhibitor, was discovered in a culture filtrate of an actinomycete. From taxonomical studies, the strain was identified and named Kitasatosporia sp. No. 55. The inhibitor was extracted with ethyl acetate, purified by Sephadex LH-20 and silica gel column chromatographies, and crystallized from methanol. The yield was about 100 mg from 18.2 I of culture filtrate. The structure of tyrostatin was N-isovaleryl-tyrosyl-Ieucyl-tyrosinal. Tyrostatin strongly inhibited pepstatin-insensitive carboxyl proteinases originating from Pseudomonas sp. No. 101 and Xanthomonas sp. No. T-22, but not any of the other pepstatininsensitive carboxyl proteinases tested. It inhibited all of the carboxyl proteinases tested of the pepstatin-sensitive group, although the inhibitory activities were not so potent. It also strongly inhibited such cysteine proteinases as papain.