The trophozoite antigens of Giardia lamblia to which host humoral and cellular immune responses are directed have not been identified. Therefore, we initiated studies to characterize these antigens in strains of G. lamblia from Afghanistan, Oregon, Ecuador, and Puerto Rico. By polyacrylamide gel electrophoresis, the electrophoretic mobility patterns of proteins of the four strains were smiliar; molecular weights of protein bands ranged between 12,000 and 140,000. The antigens which reacted with rabbit anti-G. lamblia antisera by immunoelectrophoresis were also similar for the four strains. However, comparison by crossed immunoelectrophoresis showed the Oregon strain, which has been the longest in culture, lacked a set of anodic antigens and the single neutral antigen which were present in the other three strains. In addition, other minor antigen differences between the strains were detected by this technique. When we employed trophozoites from each strain as antigen in an enzyme-linked immunosorbent assay against 10 human antisera of various titers, we also detected some differences between the strains. Although polyacrylamide gel electrophoresis and immunoelectrophoresis revealed gross similarity among G. lamblia from widely differing geographic locations, subtle differences detected by crossed electrophoresis and enzyme-linked immunosorbent assay suggest the existence of potentially important antigenic differences among these strains.