Smooth strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii, and Shigella sonnei which form pigmented colonies (Pcr+) on Congo red agar were virulent in the Sereny test. Smooth variants unable to bind Congo red (Pcr-) were avirulent. Measurements of dye uptake from solution showed that S. dysenteriae type 1 bound the most dye, followed in order of uptake by S. flexneri, S. boydii, and S. sonnei. Using the salt aggregation test (SAT) to determine cell surface hydrophobicity, we found the same order of species. The SAT could not, however, detect differences in surface properties between Pcr+ and Pcr- pairs of isogenic smooth strains. Enteroinvasive Escherichia coli strains used in the study showed SAT and Congo red-binding properties which were similar to those of the S. flexneri strains. A direct correlation was found between pigment-binding ability and the presence of the large 140-megadalton plasmid in S. flexneri, enteroinvasive E. coli, and S. boydii but not in S. dysenteriae type 1 or S. sonnei strains. Congo red interacted with outer membranes and outer membrane proteins of S. dysenteriae type 1 but not with lipopolysaccharides. However, rough mutants of Shigella species deficient in lipopolysaccharides bound Congo red and formed pigmented colonies, showing that dye binding as a virulence assay may be misinterpreted in such cases. There was complete correlation of the Pcr+ phenotype with virulence in the smooth strains in this study, suggesting that Congo red binding can be utilized as a quick and reliable alternative to the Sereny test.