In this study, we report the characterization of cell-associated and extracellular proteinases of Bodo sp., a free-living flagellate of the family Bodonidae, order Kinetoplastida. This microorganism is an evolutionary predecessor of parasitic trypanosomatids (Simpson et al., Mol. Biol. Evol., 19:2071-2083, 2002) and it is widely distributed in fresh water, marine environments and soils. Bodo sp. was grown in a monoxenic culture of bacteria for 7 days at 28°C in brain heart infusion medium (BHI) supplemented with 10% fetal calf serum. The flagellate was purified from the feeder bacteria by pelleting the cells at 1500 g for 5 min at 4°C. The cell suspension was layered on a discontinuous Percoll gradient with 5 mL steps of 25, 50, 75 and 90% Percoll in 0.25 M sucrose in Hanks balanced salt solution (pH 7.4). The purity of the fractions was assessed in Giemsa-stained smears. After centrifugation, 10⁸ cells were lysed by the addition of SDS-PAGE sample buffer. Alternatively, 10⁸ cells were incubated in PBS for 8 hours, the cells were removed by centrifugation and the supernatant was concentrated against polyethyleneglycol before the addition of sample buffer. The proteolytic activity was determined by SDS-PAGE containing co-polymerized gelatin as substrate. The SDS-PAGE-Gelatin analysis revealed that the proteolytic activity was better detected at 28°C in pH 5.5, although there was no qualitative difference among the different pH values and temperatures tested. Four cell-associated bands migrating at 120 kDa, 100 kDa, 80 kDa and 70 kDa were detected. In the bacterial pure culture, obtained by plating in BHI-Agar, only the 100 kDa band was observed. The higher molecular mass proteases belong to the serine-proteinase class, based on the inhibition by PMSF and total activity in the presence of E-64, 1,10 phenanthroline and pepstatin. Both the bacteria and the protozoan released a 100 kDa band, which was inhibited by PMSF. The biochemical characterization of proteases in non-parasitic kinetoplastid flagellates may help to determine their relationship to the already characterized trypanosomatid proteases. This approach would allow inferring an evolutionary origin of these molecules, some of which are important virulence factors in the trypanosomatids.