The causes of degradation of aquatic systems are often complex and stem from a variety of human influences. Comprehensive, multimetric biological indices have been developed to quantify this degradation and its effect on aquatic communities, and measure subsequent recovery from anthropogenic stressors. Traditionally, such indices have concentrated on small-to medium-sized streams. Recently, however, the Ohio River Fish Index (ORFIn) was created to assess biotic integrity in the Ohio River. The goal of the present project was to begin developing a companion Ohio River multimetric index using benthic macroinvertebrates. Hester–Dendy multiplate samplers were used to evaluate benthic macroinvertebrate assemblages in relation to a gradient of water quality disturbance, represented by varying distances downstream of industrial and municipal wastewater outfalls in the Ohio River. In August 1999 and 2000, samplers were set every 100 m downstream of outfalls (12 outfalls in 1999, 22 in 2000) for 300–1000 m, as well as at upstream reference sites. Candidate metrics (n = 55) were examined to determine which have potential to detect changes in water quality downstream of outfalls. These individual measures of community structure were plotted against distance downstream of each outfall to determine their response to water quality disturbance. Values at reference and outfall sites were also compared. Metrics that are ecologically relevant and showed a response to outfall disturbance were identified as potentially valuable in a multimetric index. Multiple box plots of index scores indicated greater response to outfall disturbance during periods of low-flow, and longitudinal river-wide trends. Evaluation of other types of anthropogenic disturbance, as well as continued analysis of the effects of chemical water quality on macroinvertebrate communities in future years will facilitate further development of a multimetric benthic macroinvertebrate index to evaluate biotic integrity in the Ohio River. Copyright © 2007 John Wiley & Sons, Ltd.