Concentrated animal feeding operations (CAFO) and fertilizer application to row crops may contribute to poor water quality in surface waters. To test this hypothesis, we evaluated nutrient concentrations and fluxes in four Eastern Iowa watersheds sampled between 1996 and 2004. We found that these watersheds contribute nearly 10% of annual nitrate flux entering the Gulf of Mexico, while representing only 1.5% of the contributing drainage basin. Mass budget analysis shows streamflow to be a major loss of nitrogen (18% of total N output), second only to crop harvest (63%). The major watershed inputs of nitrogen include applied fertilizer for corn (54% of total N input) and nitrogen fixation by soybeans (26%). Despite the relatively small input from animal manure (approximately 5%), the results of spatial analysis indicate that row crop and CAFO densities are significantly and independently correlated to higher nitrate concentration in streams. Pearson correlation coefficients of 0.59 and 0.89 were found between nitrate concentration and row crop and CAFO density, respectively. Multiple linear regression analysis produced a correlation for nitrate concentration with an R2 value of 85%. High spatial density of row crops and CAFOs are linked to the highest river nitrate concentrations (up to 15 mg/L normalized over five years).