Sialidases (EC 3.2.1.18) are commonly found in viruses, bacteria, fungi, protozoa, and vertebrates, but not in invertebrates. We have previously reported the presence of a new sialidase activity in the gut of exclusively hematophagous insects of the Triatoma genus, which transmit Chagas' disease (Amino, R., Acosta, A., Morita, O. M., Chioccola, V. L. P., and Schenkman, S. (1995) Glycobiology 5, 625-631). Here we show that this sialidase is present in the salivary gland of Triatoma infestans, and it is released with the saliva during the insect bite. The sialidase was purified to homogeneity (>5000 times) to a specific activity of more than 20 units/mg. It elutes from a gel filtration column with a volume corresponding to the size of 33 kDa, and it migrates as a single 26-kDa band in SDS-polyacrylamide gel electrophoresis, which is unusually smaller when compared with other known sialidases. T. infestans sialidase hydrolyzes preferentially alpha2-->3-linked sialic acids at pH 4-8, with maximal activity between pH 5.5 and 6.5, which is compatible with the optimal pH of secreted sialidases. The sialidase is competitively inhibited by 2-deoxy-2, 3-dehydro-N-acetyl-neuraminic acid (Ki = 0.075 mM) and differently from many sialidases, with exception of Salmonella typhimurium sialidase, it is inhibited competitively by HEPES (Ki = 15 mM). The fact that T. infestans sialidase is released with the saliva and can hydrolyze sialyl-LewisX blood groups, which are the ligands for selectins, suggests that it might have a role in the blood feeding.