Enterotoxigenic Bacteroides fragilis strains associated with childhood diarrhea produce a 20-kDa protein toxin (BFT). Purified BFT causes striking morphologic changes in subconfluent human colonic epithelial cells (HT29/C1). In a 3-h HT29/C1 cell assay, the estimated half-maximal effective concentration of BFT was 12.5 pM, and morphologic effects were detectable as early as 30 min and nearly complete by 1.5 h. Concentrations as low as 0.5 pM could also cause intoxication, but morphologic changes were detectable only when the assay was extended to 18 h. The onset of this intoxication was concentration dependent and rapid, occurring within minutes (<7 min at 0.25 nM, <2 min at 2.5 nM). Notably, the onset of intoxication at 37 degrees C became irreversible to washing within 2 min after exposure to BFT. Morphologic changes were completely inhibited by treatment of HT29/C1 cells with BFT at 4 degrees C but could be demonstrated by subsequent warming to temperatures of 15 degrees C or higher after washing. The time required for the association of BFT with HT29/C1 cells at 4 degrees C was inversely correlated with concentration. Inhibitors of endosomal and Golgi trafficking (NH4Cl and brefeldin A) prevented the intoxication of HT29/C1 cells by Clostridium difficile toxin A and cholera toxin, respectively, but not by BFT. Agents altering microtubule structure did not affect the cellular activity of BFT. These data indicate that a purified toxin from B. fragilis strains associated with diarrhea rapidly and irreversibly intoxicates human intestinal epithelial cells (HT29/C1) in a concentration- and temperature-dependent manner and that the process of intoxication may not involve internalization mechanisms utilizing microtubules or sensitive to pH or brefeldin A.