Quantitative biological assessment protocols are needed for monitoring river status and evaluating river rehabilitation efforts. We conducted a standardized macroinvertebrate survey at 100 sites on 38 nonwadeable rivers in Wisconsin to construct, test, and apply an index of biotic integrity (IBI) intended to be such a bioassessment tool. We assigned independent samples to IBI development (n  =  75) and IBI validation (n  =  25) data sets. We placed Hester–Dendy artificial substrates at the sites for 6 wk and processed the samples of colonizing macroinvertebrates in the laboratory with a 500-target subsampling procedure plus a large–rare taxon search. Independent of the biota, we assigned an environmental disturbance score to each site based upon water chemistry, land cover, flow modification, and point-source pollution. Ten metrics that represent macroinvertebrate assemblage structure, composition, and function constitute the IBI: the number of taxa in: 1) Insecta or 2) Ephemeroptera, Plecoptera, Trichoptera (EPT); % individuals that were: 3) Insecta, 4) intolerant EPT, 5) tolerant chironomids, 6) gatherers, 7) scrapers, or from 8) the dominant 3 taxa; 9) the mean pollution tolerance value; and 10) the number of unique ecological functional trait niches. Analyses on both the validation subset of sites and all sites inclusive confirmed that least-disturbed sites had the highest IBI scores, severely disturbed sites had the lowest scores, and moderately disturbed sites had intermediate scores. Chironominae and Hydropsychidae taxa known to tolerate nutrient enrichment and overall degraded conditions dominated samples with low IBI scores. In contrast, a diverse assemblage that thrives in relatively undisturbed conditions was present in samples with high IBI scores. Comparison of the new macroinvertebrate IBI with an existing fish IBI suggested that the indices respond to different environmental stressors and illustrated the limitations of using only one taxonomic group for bioassessment. We discuss new macroinvertebrate methods, an IBI development process, and the refinement of metrics that may be useful in tailoring assessment tools for large rivers or wadeable streams in other regions. We also present applications of the IBI, including its potential use in comprehensive large river monitoring programs and for evaluating management efforts.