A ubenimex-sensitive aminopeptidase B-like enzyme was purified from the non-membrane-bound fraction of K562 cells by a series of chromatographic procedures and slab-gel electrophoresis. The apparent molecular mass of the enzyme was estimated to be 73 kDa by SDS-PAGE. The aminopeptidase activity was activated by chloride ions and inhibited by Zn2+, Cu2+, Cd2+, and p-chloromercuribenzoic acid. Ubenimex was a potent inhibitor of this aminopeptidase in the nanomolar range. The sequence of the N-terminus of the protein was not determined. Partial amino acid sequencing revealed that the N-terminus of this aminopeptidase B-like enzyme was blocked by acylation. The partial sequences of the two fragments produced by CNBr cleavage and an acylamino acid-releasing reaction showed this enzyme to be a new aminopeptidase.