In this study, we performed fliC PCR-restriction fragment length polymorphism (RFLP) to investigate whether this technique would be better than classic serotyping for the characterization of the H antigen in enterotoxigenic Escherichia coli (ETEC) strains. We showed that the fliC genes from ETEC strains can be characterized by restriction analysis of their polymorphism. Only one allele of the fliC gene from ETEC strains was found for each flagellar antigen, with the exception of H21. Nonmotile strains could also be characterized using this molecular technique. Moreover, determination of the somatic antigen was guided by the identification of the flagellar antigen from previously unknown serotypes of ETEC strains by PCR-RFLP, thus reducing the number of anti-antigen O sera used. The PCR-RFLP technique proved to be faster than classic serotyping for the characterization of the E. coli H antigen, taking 2 days to complete instead of the 7 or more days using classic serotyping. In conclusion, the H molecular typing for Enterobacteriaceae members may become an important epidemiological tool for the characterization of the H antigen of E. coli pathotypes. The PCR-RFLP technique is capable of guiding the determination of the H antigen and could partially replace seroagglutination. With the determination of the molecular profiles of alleles from strains obtained in epidemiological studies, new patterns will be described for ETEC strains or other E. coli pathotypes, thus permitting widespread use of this technique to characterize fliC genes and determine the H antigen of E. coli strains.