We determined the surface-associated proteolytic activity in three Entamoeba histolytica Schaudinn, 1903 strains (monoxenic HM1, axenic HM1, and HK9) of known virulence and its relationship with collagenase activity. Both activities were also determined in axenic HM1 amoebae trophozoites which were sensitive and resistant to complement-mediated lysis. Surface proteolytic activity was determined in glutaraldehyde-fixcd E. histolytica trophozoites, which degraded the insoluble substrate, hide powder azure, and cleaved the human immunoglobulin G heavy chain in a time-dependent fashion, at neutral pH, in presence of 2-mercaptoethanol as cysteine protcase activator. Surface proteolytic activity was strain dependent: monoxenic HM1 > axenic IIM1 > axenic HK9. This activity correlated with collagenolytic activity (p < 0.05). Acquisition of resistance to complement-mediated lysis by axenic HM1 strain did not modify either surface proteases or collagenase expression. Our results suggest that this surface proteolytic activity could be used as an in vitro virulence marker for E. histolytica.