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A fully active derivative of ficin was prepared by reversible blocking of its active thiol group with sodium tetrathionate and by oxidation of a methionine residue with sodium metaperiodate. Treatment of this active derivative with bromoacetone in the presence of 2 M urea, pH 6.5, led to its complete inactivation, and the sole change in its composition was the loss of one of its two histidine residues. A comparison of the peptide maps of pepsin digests of the bromoacetone-treated enzyme with its untreated control revealed the disappearance of one of the three histidine-containing peptides present in the control. This particular peptide was shown to have the sequence His-Ala-Val-Ala which is identical to the sequence around the histidine which can be cross-linked to the active thiol of ficin with dibromoacetone (Husain, S. S. and Lowe, G. (1970), Biochem. J. 117, 341). During the course of these studies a very sensitive assay for measuring the activity of ficin was developed which employs p-nitrophenyl-N-alpha-carbobenzoxy-L-citrullinate as the substrate.