The infective larvae of Necator americanus were shown to secrete all mechanistic classes of proteolytic enzymes with two overall pH optima of 6.5 and 8.5 using fluorescein isothiocyanate-labeled casein as the substrate. Since infective larvae are obligate skin penetrators, the effect of each of these enzyme classes against macromolecules derived from human skin was examined. Larval secretions were shown to degrade collagen types I, III, IV, and V, fibronectin, laminin, and elastin. All the skin macromolecules tested were hydrolyzed by aspartyl proteinase activity, which was inhibitable by pepstatin A. Collagen and elastin was also hydrolyzed by metalloproteinase activity, while the serine proteinase activity hydrolyzed only elastin. As a consequence of these experiments, the effect of proteinase inhibitors on the penetration of live larvae through hamster skin was tested. Larval penetration was significantly inhibited only by pepstatin A, confirming the importance of the aspartyl proteinase activity during the skin penetration process.