This is a quantitative study of hydrolysis of the His-Gly bond in the peptide AcHis-Gly catalyzed by cis-[Pd(en)(H2O)(2)](2+). We exploit the diverse coordinating abilities and acid-base properties of histidyl residue to interpret the kinetics and explain the mechanism of this new reaction. We compare peptides selectively methylated at the N-1 or N-3 atom of imidazole and study effects of solution acidity on the abundance of different pepride-catalyst complexes and on the late constant for hydrolysis. Only the catalyst bound to the N-3 atom of imidazole can effect this reaction; none of the four other modes of coordination is effective. The necessary approach of the palladium(II) aqua complex to the scissile peptide bond and the rate constant of hydrolysis are unaffected by the remote methyl group that merely controls the mode of peptide coordination to the catalyst. Acid in solution affects hydrolysis only by controlling the concentration of the reactive complex, not by catalyzing the reaction itself. Weakly acidic solution is required to suppress oligomerization of the catalyst. Hydrolytic cleavage occurs with a turnover greater than 4. With the halflife of 5.1 h at pH 5.0, the cleavage is fast enough at relatively mild conditions to be practical for various applications in biochemistry and structural biology. This study is an important step in our development of palladium(TI) complexes as artificial metallopeptidases.