The use of fish communities to assess environmental quality is common for streams, but a standard methodology for large rivers is as yet largely undeveloped. We developed an index to assess the condition of fish assemblages along 1,580 km of the Ohio River. Representative samples of fish assemblages were collected from 709 Ohio River reaches, including 318 "least-impacted" sites, from 1991 to 2001 by means of standardized nighttime boat-electrofishing techniques. We evaluated 55 candidate metrics based on attributes of fish assemblage structure and function to derive a multimetric index of river health. We examined the spatial (by river kilometer) and temporal variability of these metrics and assessed their responsiveness to anthropogenic disturbances, namely, effluents, turbidity, and highly embedded substrates. The resulting Ohio River Fish Index (ORFIn) comprises 13 metrics selected because they responded predictably to measures of human disturbance or reflected desirable features of the Ohio River. We retained two metrics (the number of intolerant species and the number of sucker species [family Catostomidae]) from Karr's original index of biotic integrity. Six metrics were modified from indices developed for the upper Ohio River (the number of native species; number of great-river species; number of centrarchid species; the number of deformities, eroded fins and barbels, lesions, and tumors; percent individuals as simple lithophils; and percent individuals as tolerant species). We also incorporated three trophic metrics (the percent of individuals as detritivores, invertivores, and piscivores), one metric based on catch per unit effort, and one metric based on the percent of individuals as nonindigenous fish species. The ORFIn declined significantly where anthropogenic effects on substrate and water quality were prevalent and was significantly lower in the first 500 m below point source discharges than at least-impacted sites nearby. Although additional research on the temporal stability of the metrics and index will likely enhance the reliability of the ORFIn, its incorporation into Ohio River assessments still represents an improvement over current physicochemical protocols.