After acute immunoreactive infestation with the Chagas' disease parasite, Trypanosoma cruzi, some patients develop chronic megacolon, whereas others remain asymptomatic. Chronic chagasic patients with gastrointestinal involvement exhibit inflammation and degeneration of enteric neurons. Our hypothesis is that enteric glial cells may be involved in the modulation of enteric inflammatory responses or even control the colon's dilatation. The aims of this study were to characterize the phenotype of enteric glial cells according to the expression of S-100 and glial fibrillary acidic protein and to look for correlation between these data and the neuronal loss in the colon of chagasic patients. We studied both dilated and nondilated portions of chagasic megacolon. We used a pan-enteric glial cell marker (anti-S-100), a subpopulation enteric glial cell marker (anti-glial fibrillary acidic protein), and a pan-neuronal marker (anti-Human protein C and protein D) with double-labeled sheets using a confocal microscope. Our results demonstrate that neuronal loss is similar in dilated and nondilated portions of chagasic megacolon. Moreover, the results indicate that neuronal destruction present in chagasic megacolon is preceded by glial component loss. The nondilated portion of chagasic megacolon exhibited increased expression of glial fibrillary acidic protein comparable with the dilated portion and also to the noninfected group. Our results suggest that glial fibrillary acidic protein enteric glial cells prevent dilatation of the organ and protect the enteric nervous system against the inflammatory process and neuronal destruction, preventing the destruction from expanding to unaffected areas of the colon.