Abstract Almeida-de-Faria, M., Freymüller, E., Colli, W., and Alves, M. J. M. 1999. Trypanosoma cruzi: Characterization of an intracellular epimastigote-like form. Experimental Parasitology92, 263–274. A detailed study of transient epimastigote-like forms as intermediates in the differentiation of Trypanosoma cruzi amastigotes to trypomastigotes inside the host cell cytoplasm was undertaken using the CL-14 clone grown in cells maintained at 33°C. Several parameters related to these forms have been compared with epimastigotes and other stages of the parasite. Consequently, the designation of intracellular epimastigotes is proposed for these forms. Despite being five times shorter (5.4 ± 0.7 μm) than the extracellular epimastigote (25.2 ± 2.1 μm), the overall morphology of the intracellular epimastigote is very similar to a bona fide epimastigote, when cell shape, position, and general aspect of organelles are compared by transmission electron microscopy. Epimastigotes from both sources are lysed by human complement and bind to DEAE–cellulose, in contrast to amastigotes and trypomastigote forms. A monoclonal antibody (3C5) reacts with both epimastigotes either isolated from axenic media or intracellular and very faintly with amastigotes, but not with trypomastigotes. Some differences of a quantitative nature are apparent between the two epimastigote forms when reactivities with lectins or stage-specific antibodies are compared, revealing the transient nature of the intracellular epimastigote. The epitope recognized by 3C5 monoclonal antibody reacts slightly more intensely with extracellular than with intracellular epimastigotes, as detected by immunoelectron microscopy. Also a very faint reaction of the intracellular epimastigotes was observed with monoclonal antibody 2C2, an antibody which recognizes a glycoprotein specific for the amastigote stage. Biological parameters as growth curves in axenic media and inhability to invade nonphagocytic tissue-cultured cells are similar in the epimastigotes from both origins. It is proposed that the epimastigote-like forms are an obligatory transitional stage in the transformation of amastigotes to trypomastigotes with a variable time of permanency in the host cell cytoplasm depending on environmental conditions.