Abstract: Urbanization represents a strong and increasingly more prevalent impact on stream quality worldwide. One of the characteristic effects of increased urbanization is a consistent decline in biological stream condition. The characterization of this biological degradation with increasing urbanization presents a number of advantages for the study and management of urban streams and catchments. In this paper, the limitation of biological condition with urbanization, called observed biological potential, is characterized. Using an urban intensity index and a biological index developed specifically for urban systems in the Baltimore, Maryland; Cleveland, Ohio; and San Jose, California regions, two principal techniques were compared (quantile regression and bin regression) to define observed biological potential along urban gradients. Quantile regression was selected as the preferable tool for describing observed biological potential given the consistency with which it can be applied and its statistical efficiency, however, bin quantile regression performed similarly. Having identified a numeric approximation of observed biological potential, two methods for identifying factors related to distance from potential as a way of identifying critical environmental factors affecting biological condition in urban areas were explored. The results of this work can be used for identifying benchmarks for urban stream biological condition, identifying limiting catchment characteristics, and prioritizing urban stream management efforts.