Enterotoxigenic Escherichia coli strains were divided into five groups on the basis of their bacterial surface hydrophobicity (Honda et al., FEMS Microbiol. Lett. 17:273-276, 1983). Strains in group III showed heat-stable high hydrophobicity, although they did not show mannose-resistant hemagglutination with either human or bovine erythrocytes. E. coli strain 260-1 in group III was characterized. Electron microscopic examination revealed the presence of pili on the surface of this strain, but not on that of strain 260-1a, which is a mutant of 260-1 showing low hydrophobicity. When strain 260-1 was grown at 18 degrees C, it did not produce pili or show high hydrophobicity. On homogenization of strain 260-1 grown at 37 degrees C the high hydrophobicity and the pili on its surface were lost. These results indicate that the pili of strain 260-1 are associated with the hydrophobicity. Strain 260-1 pili were purified to homogeneity by successive column chromatographies on Sepharose 4B and phenyl-Sepharose CL-4B. Their molecular weight was estimated to be about 18,000. An antigenic difference between purified pili of strain 260-1 and colonization factor antigens I and II was demonstrated. The colonization ability of E. coli 260-1 was shown by animal experiments on suckling mice and infant rabbits. From these results it is concluded that the pili of strains in group III of human enterotoxigenic E. coli, which may play a role in colonization, are of a new type.