Palladium(II) ions anchored to side chains of histidine and methionine residues in peptides and proteins in weakly acidic aqueous solutions promote hydrolytic cleavage of proximate amide bonds in the backbone. In this study, we determine how attachment of Pd(II) ions to histidine and methionine anchors and also to the terminal amino group in six natural peptides (chains A and B of insulin, segment 11-14 of angiotensinogen, pentagastrin, angiotensin 11, and segment 3-8 of angiotensin 11) and two proteins (ubiquitin and cytochrome c) affects regioselectivity and rate of backbone cleavage. These Pd(II)-promoted reactions follow a clear pattern of regioselectivity, directed by the anchoring side chains. When the Pd(II) reagent is nonspecifically anchored to the terminal amino group, the ligating site that is present in almost all proteins, the cleavage is fortunately absent. When the reagent is anchored to a residue in positions 1, 2, or 3, cleavage is absent, because the terminal amino group and deprotonated amide nitrogen atom(s) interposed between it and the anchor "lock" the Pd(II) ion in hydrolytically inactive chelate complexes. When the reagent is anchored to residues in positions beyond 3, the second amide bond upstream from the anchor is regioselectively cleaved in all cases when the anchor was "isolated," that is, flanked by noncoordinating side chains. Segment 3-8 of angiotensin 11 undergoes additional cleavage, which we explain by determining the rate constants for the cleavage, identifying the rate-limiting displacement of ethylenediamine ligand from the Pd(II) ion, and detecting several intermediates. Experiments with cytochrome c demonstrate that the number of cleavage sites can be controlled by adjusting the mole ratio of the Pd(II) reagent to the substrate. Our inorganic peptidases are useful for biochemical applications because their regioselectivity and reactivity set them apart from proteolytic enzymes and organic chemical reagents.