We used the polymerase chain reaction (PCR) to study the epidemiology of pathogenic and nonpathogenic Entamoeba histolytica in a rural community in Mexico. Formalin-fixed stool samples were used for extraction of DNA. The PCR amplifications were performed using two sets of primers that discriminate between pathogenic or non-pathogenic E. histolytica. A total of 201 randomly selected individuals were studied. Among them, 25 (12%) were diagnosed to be infected with E. histolytica by microscopy; PCR identified 24 of these as positive (sensitivity = 0.96) and of 176 negative individuals, only three were identified as positive (specificity = 0.98). The PCR analysis defined three populations: 14 cases were positive for both pathogenic and nonpathogenic E. histolytica, nine cases were positive for pathogenic and negative for nonpathogenic E. histolytica, and only one case was negative for pathogenic and positive for nonpathogenic E. histolytica. Infection by E. histolytica was strongly associated to infection with Entamoeba coli (odds ratio [OR] = 9.41, 95% confidence interval [CI] = 3.09, 28.65, P < 0.0004) and Endolimax nana (OR = 6.15, 95% CI = 2.03, 18.17, P < 0.0004). This new technique has high specificity and sensitivity; it is simple, reproducible, fast, avoids the need to culture trophozoites, and can be applied in the field for epidemiologic studies.