A total of twenty-five norfloxacin resistant Escherichia coli were isolated from Joongrang-chun stream, a branch of the Han River in Seoul, Korea from May to July in 2000 and their norfloxacin resistance mechanism was characterized for target site mutation, permeability, and efflux pump. Fourteen isolates contained the same three mutations, Ser83->Leu and Asp87->Asn in GyrA and Ser90->Ile in ParC. Six isolates had Ser83->Leu and Asp87->Tyr in GyrA and Ser80->Ile in ParC while one isolate had Ser83->Leu and Val103->Ala in GyrA and Ser80->Ile in ParC. Two isolates had mutation(s) in GyrA without any mutation in ParC. Two isolates had Ser80->Arg in ParC instead of the commonly found Ser80->Ile. Every norfloxacin resistant isolate had an efflux system but the correlation between the efflux activity and MIC was not observed. The amount of OmpF for norfloxacin permeability decreased in resistant isolates compared to the susceptible strains. When amplified polymorphic DNA (RAPD) and pulse field gel electrophoresis (PFGE) were performed, these isolates showed no similarity to each other or clinical isolates.