Comparison of proteolytic enzymes between different insect orders had provided information about specificity and evolution. However, a comparison of evolutionary trends of Arthropoda peptidases is not possible due to the lack of data mainly for Arachnida enzymes. In order to understand protein digestion in spiders the digestive juice (DJ) and the hepatopancreas (HP) from adult female Nephilengys cruentata (giant spider) were characterized. The following enzymes were measured in both samples, HP and DJ, respectively: aminopeptidase (Leu-p-nitroanilide; 2,4 mU/mg; 3,0 mU/mg), dipeptidase (Gly-Leu, 1,4 mU/mg; 2,0 mU/mg); carboxypeptidase (Carbobenzoxy-GlyPhe 3,0mU/mg; 0,3 mU/mg), endopeptidase (Carbobenzoxy-PheArg-7-amido-4-methyl-coumarin 11U/mg; 6,7 U/mg). Cysteine-proteinase has an optimum pH of 5,9. Inhibition assays indicated that the major endopeptidase activity is a cysteine proteinase in both samples (60% of inhibiton by E-64 HP and 100% of inhibition on DJ. PMSF had no effect on this activity). Western and dot blotting experiments also indicated the presence of cysteine proteinase in N. cruentata. Both samples were submitted to gel filtration and the cysteine proteinase activity from both DJ and HP presented the same molecular mass (57kDa, determined by gel filtration). Western and dot blotting experiments confirmed the presence of aminopeptidase, which presented optimum pH of 7.3 (HP) and 7.9 (DJ). Ion-exchange chromatographies indicated that carboxypeptidase and dipeptidase do not differ between both samples, suggesting that the hepatopancreas is the site of their secretion. On the other hand, cysteine proteinase activity presented one activity in digestive juice and at least two activities in hepatopancreas.