A novel protease exhibiting substrate specificity toward paired basic residues has been partially purified from the soluble fraction of bovine adrenal chromaffin granules by utilizing an affinity chromatography on STI-Sepharose. The enzyme, with optimal pH around 7.5-9.5, is classified into a serine-protease by its inhibitor spectrum. The enzyme specifically cleaved the Lys-Arg bonds of two synthetic peptides containing the subsequence of proenkephalin A, but endogenous opioid peptides containing a single basic residue in the molecule [Met)enk-Arg-Phe, (Met)enk-Arg-Gly-Leu) were not affected by the enzyme. The unique substrate specificity of the enzyme, which is well in accord with the processing pattern of proenkephalin A in adrenal medulla, indicates that the enzyme may be physiologically involved in proenkephalin processing.