Enzymes with gelatinolytic activity were detected in Tityus bahiensis and Tityus serrulatus venom. Their activity was optimal at pH 8.0 in SDS–PAGE–gelatin. They were inhibited by PMSF but not by iodoacetamide, pepstatin or phenantrolin in the assay conditions used. This suggests that these enzymes are serine proteases. The presence of metal ions did not affect the proteolytic activity of these enzymes. Several possible functions may be envisaged for these enzymes: in tissue permeabilization, pancreatitis and toxin processing.