The bacterial flora of the intestine plays an important role in the virulence caused by Entamoeba histolytica. Cysteine proteinase (CP), an amoebic virulence factor, plays a major role in host cell destruction. The mechanism of increased virulence following bacterial co-association is not understood. We studied CP of E. histolytica HM1:IMSS which was co-associated with Escherichia coli K12 strain pre-incubated with GalNAc or CP specific inhibitor E 64. Co-association of E. histolytica with bacteria enhanced CP activity 3-6-fold as assessed by azocasein assay and substrate gel electrophoresis showed bands at molecular weights of 28, 35 and 56 kDa. Northern and Western blot analysis showed increase in ehcp2 and ehcp5 gene expression. Trophozoites co-associated with E. coli showed greater cytotoxicity of BHK cells by a 51Cr release assay than trophozoites that had not been co-associated; this enhancement was abolished by E-64 treatment. The killing of BHK 21 targets by E. histolytica was characterized by DNA laddering which was not inhibited with E-64. GalNAc pre-incubation of trophozoites reduced cytotoxicity and DNA laddering, while E. coli co-associated E. histolytica showed smearing with faint laddering of BHK implicating both necrosis and apoptosis. Hence, bacterial co-association increases CP activity and CP gene expression and contributes to the necrosis of the target cell.