Preservation of extraordinary natural resources, protection of water quality, and restoration of impaired waters require a strategy to identify and protect least-disturbed streams and rivers. We applied two objective, quantitative methods to determine stream ecological integrity of headwater reaches of 10 Ozark rivers, 5 with Wild and Scenic River federal protective status. Thirty-four variables representing macroinvertebrate and fish assemblage characteristics, in-stream habitat, riparian vegetation, water quality, and watershed attributes were quantified for each river and analyzed using two multivariate approaches. The first approach, cluster and discriminant analyses, identified two groups of river with only one variable (% forested watershed) reliably distinguishing groups. Our second approach employed ordinal scaling to compare variables for each river to conceptually ideal conditions that were developed as a composite of optimal attributes among the 10 rivers. The composite distance of each river from ideal was then calculated using a unidimensional ranking technique. Two rivers without Wild and Scenic River designation ranked highest relative to ideal (highest ecological integrity), and two others, also without designation, ranked most distant from ideal (lowest ecological integrity). Fish density, number of intolerant fish species, and invertebrate density were influential biotic variables for scaling. Contributing physical variables included riparian forest cover, water nitrate concentration, water turbidity, percentage of forested watershed, percentage of private land ownership, and road density. These methods provide a framework for refinement and application in other regions to facilitate the process of establishing least-disturbed reference conditions and identifying rivers for protection and restoration. ?? 2005 Springer Science+Business Media, Inc.