Epsilon toxin (∊-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant ∊-toxin–green fluorescence protein (∊-toxin–GFP) and ∊-prototoxin–GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant ∊-toxin–GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. ∊-Toxin–GFP and ∊-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with ∊-toxin–GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that ∊-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an ∊-toxin receptor is expressed on renal distal tubules of mammalian species, including human. (J Histochem Cytochem 52:931–942, 2004)