Flagellin (fliC) genes of 12 Shigella boydii and five Shigella dysenteriae strains were characterized. Though these strains are nonmotile, the cryptic fliCSB gene, cloned from S. boydii strain C3, is functional for expression of flagellin. It consists of 1,704 bp, and encodes 568 amino acid residues (57,918 Da). The fliCSD gene from S. dysenteriae strain 16 consists of 1,650 bp encoding 549 amino acid residues (57,591 Da) and contains an IS1 element inserted in its 3' end. The two genes are composed of the 5'-constant, central variable and 3'-constant sequences, like other known fliC genes. The two genes share high homology in nucleotide and amino acid sequences with each other and also with the Escherichia coli fliCE gene, indicating that both genes are closely related to the fliCE gene. Comparison of the central variable sequences of six different fliC genes showed that the fliCSB and fliCSD genes share low homology in amino acid sequence with the other fliC genes, suggesting that they encode antigenic determinants intrinsic to respective subgroups. However, Southern blotting using as probes the central variable sequences of several fliC genes showed that four of 12 S. boydii strains have a fliC gene similar to that of Shigella flexneri, and that among five fliC genes from S. dysenteriae strains, one is similar to that of S. flexneri, two are similar to that of S. boydii, and only one is unique to S. dysenteriae. Some of these variant alleles were verified by immunoblotting with flagellins produced from cloned fliC genes. The presence of variant fliC alleles in S. boydii and S. dysenteriae indicates that subdivision into subgroups does not reflect the ancestral flagella H antigenic relationships. These data will be useful in considering the evolutionary divergence of the Shigella spp..