A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.