Blood-feeding parasites use mechanistically distinct proteases to digest hemoglobin (Hb), often as multienzyme cooperative cascades. We investigated the roles played by 3 distinct proteases from adults of the human hookworm Necator americanus. The aspartic protease Na-APR-1 and the cysteine protease Na-CP-3 were expressed in catalytically active form in yeast, and the metalloprotease Na-MEP-1 was expressed in catalytically active form in baculovirus. Antibodies to all 3 proteases were used to immunolocalize each native enzyme to the intestine of adult N. americanus. Recombinant Na-APR-1 cleaved intact human Hb. In contrast, Na-CP-3 and Na-MEP-1 could not cleave Hb but instead cleaved globin fragments that had been hydrolyzed by Na-APR-1, implying an ordered process of hemoglobinolysis. Seventy-four cleavage sites within Hb alpha- and beta-chains were characterized after digestion with all 3 proteases. All of the proteases demonstrated promiscuous subsite specificities within Hb; noteworthy preferences included aromatic and hydrophobic P1 residues and hydrophobic P1' residues for Na-APR-1 and hydrophobic P1 residues for Na-MEP-1. We conclude that Hb digestion in N. americanus involves a network of distinct proteases, some of which act in an ordered fashion, providing a potential mechanism by which some of these hemoglobinases exert their efficacy as recombinant vaccines against hookworm infection.