A reproducible, experimental model of columnaris disease was developed to study the pathogenesis of cutaneous disease associated with Flavobacterium columnare infection in koi (Cyprinus carpio). In experimental infections, lesions were usually restricted to skin and fins; gill necrosis was not a consistent finding. Cytologic and histopathologic examinations provided a presumptive diagnosis of columnaris disease. Specific detection of F. columnare was done using the polymerase chain reaction and DNA in situ hybridization (ISH). Polymerase chain reaction allowed the detection of F. columnare in fresh biological material and in formalin-fixed, paraffin-embedded tissues. The DNA ISH technique allowed the identification and localization of F. columnare in formalin-fixed, paraffin-embedded tissues. Using these molecular techniques, F. columnare was readily detected in skin specimens from infected fish; however, the bacterium was infrequently detected in specimens of liver, kidney, and spleen. These observations suggest that columnaris disease generally presents as a cutaneous disease that is unassociated with systemic infection in koi. Hematologic studies indicated that most infected koi developed microcytic, normochromic, nonregenerative anemia and leukopenia characterized by lymphopenia, mild neutrophilia, and monocytosis. Biochemical changes in diseased fish included significant hyperglycemia, hyponatremia, and hypochloridemia.