The index of biotic integrity (IBI) has been used to assess the biological quality of flowing water systems in areas throughout the United States. Yet, only rarely has biotic integrity been related to independent measures of water or habitat quality. We show that the IBI ranks sites similarly in two Illinois watersheds where conditions remained relatively stable during 3 years of sampling. Further, rankings among sites conform to prior assessments of site quality based on habitat and water quality. Neither a species diversity index (H') nor any of the individual metrics that constitute IBI performed as consistently at ranking sites as did the IBI. Sampling should be conducted during early summer to reduce variation due to seasonal fish migration and fall recruitment of young-of-the-year fish. In an Indiana watershed subject to extensive conservation planning, the IBI reflects known habitat and water quality perturbations of both natural and anthropogenic origin. Little or no improvement in biotic integrity was detected following implementation of numerous soil and water conservation practices.