The present study analyses the morphology and the exposition of surface carbohydrates and the Ssp4 antigen of amastigote forms of Trypanosoma cruzi (Y strain) obtained from three different sources: (a) intracellular, isolated from infected Vero cells 3 days after infection, (b) extracellular, isolated from the supernatant of Vero cells 15 days after infection, and (c) axenic, obtained by incubation of tissue culture trypomastigotes in LIT medium, at 37 degrees C for 4 days. No morphological differences were observed by light microscopy among these amastigotes. Transmission electron microscopy of thin sections showed a thick cell coat easily observed on the plasma membrane of axenic amastigotes. Carbohydrate-containing sites on the surface of the three different amastigotes were analysed using lectins, agglutination assays and flow cytometry. Mannose and/or glucose residues were found on the surface of all populations, but intracellular amastigotes showed the highest number. A small group of cells from the different populations expressed galactose and N-acetyl-glucosamine residues. The presence and distribution of the Ssp4 antigen in the different amastigote populations were evaluated using FITC and gold-labelled antibodies, and observed with an electronic programmable individual cell sorter and transmission electron microscopy. Ssp4 antigen was present on the membrane lining the flagellar pocket and on the cell surface, as well as inside the cytoplasmic vesicles of the host cell. Flow cytometry analysis of different amastigote populations showed that intracellular amastigotes presented the highest percentage of Ssp4-expressing cells.