Entamoeba histolytica is an invasive intestinal amoeba morphologically indistinguishable from Entamoeba dispar, a closely related organism that is not able to invade tissues. Differential diagnosis under conventional microscopy is therefore impossible. Reliable tools are needed for clinical diagnosis and for the reevaluation of the prevalence of infection with the invasive species worldwide. Monoclonal Antibody (MAb) 20/7D exhibited promising results when ascites was used to identify cultured isolates of E. histolytica by indirect immunofluorescence assays (IFA), and when used in a Faecal Antigen Capture Enzyme-Linked Immunosorbent Assay (FAC-ELISA) for laboratory diagnosis of amoebic dysentery and colitis. Here, further development of the assay was attempted to increase its sensitivity and use it for detection of asymptomatic carriers of E. histolytica. After purification and subsequent titration in ELISA, MAb 20/7D did not adequately distinguish between crude lysates of cultured E. histolytica and E. dispar trophozoites. MAb 20/7D reacted with a similar soluble antigen of E. histolytica and E. dispar, which confirmed previous observations in western blot analysis under non-reducing conditions. Therefore, the use of the FAC-ELISA for diagnosis in areas where E. dispar is endemic is probably not viable. A nucleic acid detection method was therefore developed. Polymerase Chain Reaction was used to amplify specific tandem sequences in the 24.5 Kb episome of E. histolytica and E. dispar. After PCR, internal sequences of digoxigenin-labelled PCR products were hybridized to specific biotin-labelled probes for E. histolytica or E. dispar and detected in Enzyme- Linked Immunosorbent Assay (ELISA). The Polymerase Chain Reaction Solution- Hybridisation Immunosorbent Assay (PCR-SHELA) was evaluated on samples from travellers returning from the tropics to Barcelona. The sensitivity and specificity were 98% and 100% respectively, when results were compared with microscopy. PCR-SHELA was also useful for differential diagnosis in cases of amoebic abscesses, amoebic dysentery, salmonellosis, ulcerative colitis and in asymptomatic carriage of E. histolytica. The new test gives sensitive and specific differentiation between E. histolytica and E. dispar in clinical specimens and it has proved successful in screening faecal samples in endemic areas for epidemiological purposes.