Ribonuclease T1 [EC 2.7.7.26] was rapidly inactivated by rose bengal-catalyzed photooxidation at pH8.5 and 37°C, losing the activity toward both RNA and 2′, 3′-cyclic guanylate in parallel. The activity was lost more slowly at pH7.0 and little inactivation occurred at pH5.5, suggesting the implication of histidine residues in the inactivation. The only marked changes occurred with histidine and tryptophan residues. About 70% and over 90% inactivation, respectively, took place when one and two histidine residues per molecule of protein were lost. The tryptophan residue was lost much more slowly; the loss was only 0.2 residue per molecule of protein at the level of 80% inactivation. The reactivity of the γ-carboxyl group of the active site glutamic acid-58 toward iodoacetate was also diminished markedly. These results thus show clearly that the photooxidation of one or two histidine residues are directly responsible for the inactivation, and hence that one or both of them are involved in the active center of the enzyme.