Current diagnosis of Entamoeba histolytica infection requires the direct microscopic identification of the parasite, a technique that is insensitive and cannot distinguish pathogenic E. histolytica from noninvasive E. dispar. Enzyme-linked immunosorbent assay (ELISA) antigen detection tests were developed to distinguish E. histolytica from E. dispar infection in stool specimens. The ELISA result for E. histolytica antigen was positive in 26 of 27 E. histolytica-positive stool specimens, three of 25 E. dispar-positive stools, and one of 30 stools with other or no intestinal parasites, giving a specificity and sensitivity for the detection of E. histolytica infection of 93% and 96%, respectively. The assay result used to detect both E. dispar and E. histolytica was positive in 26 of 27 E. histolytica-positive stools, 19 of 25 E. dispar-positive stools, and one of 30 stools negative by microscopy and culture for Entamoeba, giving a specificity and sensitivity of 97% and 87%, respectively. Because these ELISAs can be completed in several hours, they offer promise as rapid and sensitive means of detecting amebic infection.