Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic alpha-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.