The prevalence and species distribution of Entamoeba histolytica and E. dispar in the Venda region were determined in stool samples collected from public hospitals and primary schools by ELISA and a nested polymerase chain reaction (PCR). E. histolytica was detected in 37/197 (18.8%) and 1/47 (2.1%) samples, whereas 50/197 (25.3%) and 4/47 (8.5%) had E. dispar in the hospitals and schools, respectively. The age groups most infected were 0-2 (33%) years followed by 20-29 years (27%). E. histolytica was significantly associated with diarrhea (77.4% versus 22.6%; chi 2 = 39.48, P < 0.05), and with the presence of lactoferrin (85.7% versus 14.2%) in the stools, indicating intestinal inflammation (chi 2 = 29.605, P < 0.05). E. histolytica was found in 5 (16.12%) of the 31 HIV-positive individuals and in 33 (15.5%) of the 213 HIV-negative individuals. E. histolytica infections are common in the Venda region and are associated with diarrhea and intestinal inflammation.