We have investigated the subcellular location of the Trypanosoma cruzi surface glycoprotein, Gp72, by introducing epitope-tagged copies of gp72 into gp72 null-mutant cells, A tagged Gp72, containing three tandemly repeated copies of a human influenza hemagglutinin nonapeptide (HA) adjacent to the mature Gp72 amino terminus, was able to complement the null mutant phenotype, as well as being recognized in western blots by both anti-HA antibody and the carbohydrate-specific monoclonal antibody WIC29.26, Integration of this epitope-tagged gp72 into the chromosomal gp72 locus produced a clonal cell line, 72HAN3.1G7, which was used for studies of the subcellular location of the epitope-tagged Gp72, Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 epimastigotes showed that Gp72 was evenly distributed over the cell body and somewhat concentrated in the proximal region of the flagellum, No fluorescence could be detected in the distal tip of the flagellum, Immunoelectron microscopy of fixed 72HAN3.1G7 epimastigotes revealed that Gp72 was predominantly membrane-associated and located on the cell surface, Indirect immunofluorescence microscopy of live 72HAN3.1G7 epimastigote cells showed a similar pattern of fluorescence on the flagellum, but no fluorescence was detected on the cell body, which was attributed to masking by other cen-surface components. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 amastigotes revealed that Gp72, which has long been considered to be expressed only in epimastigotes and metacyclic trypomastigotes, can be expressed in amastigotes, but it no longer contains the WIC29.26 carbohydrate epitope.