Proteolytic activity in excretory/secretory products (ESP) of first- (L1), second- (L2) and third-instar (L3) larvae of Dermatobia hominis was analyzed through gelatin-gel and colorimetric enzyme assays with the chromogenic substrates azocasein and BApNA. The functional characterization of proteases was based on inhibition assays including synthetic inhibitors. ESP were obtained from new-hatched larvae reared in the laboratory and from second- and third-instar larvae removed from naturally infested cattle. Gelatin-gel analysis evidenced few bands of proteolysis, predominantly of high apparent molecular masses, in ESP of L1, whereas in the gel of L2 and U ESP there was a wide range of proteolytic activity most of them not resolved in a single species. Azocasein assays revealed a progressive increase of protease activity from first- to third-instar larvae. Protease inhibitor assays revealed a predominance of metalloproteases in L1 ESP that could be related to a skin penetration process and to a diversion of host immune response. The predominance of serine proteases in L2 and L3 and the great tryptic activity presented by L3 ESP were attributed to an increasing trophic activity by the growing larvae, since the viability of adult flies strictly depends on larval abilities to assimilate nutrients from the host. Taking together, these results suggest that Dematobia larvae secrete/excrete different proteases that may be related to diverse functions during host penetration and infestation, which reinforces the relevance of the study of such proteolytic enzymes. (C) 2009 Elsevier B.V. All rights reserved.