Naturally high total dissolved solids and upstream agricultural runoff often mask the influence of urban land cover on stream chemistry and biology. We examined the influence of headwater urbanization on the water chemistry, microbiology, and fish communities of the Big Brushy Creek watershed, a 96 km 2 drainage basin in the piedmont of South Carolina, USA. Concentrations of most major anions and cations (especially nitrate, sulfate, chloride, sodium, potassium, and calcium) were highest in the urban headwaters and decreased downstream. Generally, the highest concentrations of suspended coliform bacteria occurred in the urban headwaters. In contrast, stream habitat quality and the abundance, species richness, and species diversity of fishes did not differ significantly between urban and rural sites. Discharge of wastewater treatment plant effluent at one rural location caused an increase in concentrations of many solutes and possibly the abundance of benthic algae. We hypothesize that atmospheric dry deposition and domestic animal wastes are important sources of stream solutes and of coliform bacteria, respectively, in the urban headwaters. The lack of significant differences in fish abundance and diversity between urban and rural sites may indicate that urban development in the Big Brushy Creek watershed has not yet degraded habitat conditions greatly for stream fishes. Alternatively, agriculture or other land uses may have degraded stream habitat quality throughout the watershed prior to urbanization.