The binding of small molecules (ligands) to proteins is an important event in many biological processes. One strategy to locate ligand binding sites on proteins is to attach a photochemical reagent to the ligand. Such an approach requires the design of photochemical reagents that are capable of binding proteins at specific sites. In this study, a new pyrenyl probe, d-biotinyl-1(1-pyrene)methylamide (Py–biotin) was designed and synthesized by coupling of d-biotin to 1(1-pyrene)methylamine hydrochloride. Binding studies and site-specific photocleavage of avidin and streptavidin by Py–biotin were demonstrated. Red shifts of the absorption peak positions of the pyrenyl chromophore followed by hyperchromism were observed upon binding to avidin. The photocleavage of avidin was achieved when a mixture of the protein, Py–biotin, and an electron acceptor, cobalt(III) hexammine trichloride (CoHA), was irradiated at 342 nm. N-terminal sequencing of the peptide fragments indicated a cleavage site of avidin between Thr 77 and Val 78. The formation of pyrenyl cation radical resulted from quenching of pyrene excited state by CoHA is expected to play the important role in the photoreaction. In addition, the photocleavage of protein by a molybdenum complex is also demonstrated in this report. A molybdenum(VI) peroxo α-amino acid complex, MoO(O2)2(α-leucine) (H2O), was synthesized and used for site-specific cleavage of porcine pepsin, a model protein. Photocleavage of pepsin by MoO(O2)2(α-leucine)(H2O) was achieved upon irradiation of the pepsin/MoO(O2)2(α-leucine)(H2O) mixture by UV light (320 and 340 nm) for 10-30 minutes. No cleavage was observed in the absence of MoO(O2)2(α- leucine)(H2O) or the light. The photocleavage yield increased with irradiation time. Nterminal sequencing of the cleaved fragments indicated the assigned cleavage site to Leu(112)-Tyr(113). The cleavage reaction was quenched by ethanol. Therefore, hydroxyl radicals may be involved in the reaction and responsible for the cleavage of the protein. This is the first demonstration of the successful photocleavage of proteins by a molybdenum complex. This observation can provide a new approach for the photochemical footprinting of metal binding sites on proteins.