We measured nitrification rates in sediment samples collected from a variety of aquatic habitats in Navigation Pool 8 of the Upper Mississippi River (UMR) 7 times between May 2000 and October 2001. We also conducted nutrient-enrichment experiments and analyzed vertical profiles of sediment to determine factors regulating nitrification. Nitrification rates were relatively high compared to other ecosystems (ranging from 0–8.25 μg N cm⁻² h⁻¹) and exhibited significant temporal and spatial patterns. Nitrification rates were greatest during the summer and spring compared to autumn and winter (ANOVA, p < 0.05) and were greater in contiguous backwater and impounded habitats compared to main and side-channel habitats (p < 0.05). Regression analysis indicated that nitrification rates were weakly (r² = 0.18, p < 0.0001) related to temperature and exchangeable NH₄ of the sediment. However, nutrient-enrichment experiments showed that NH₄ availability did not limit nitrification in 3 sediment types with variable organic matter. Vertical profiles of sediment cores demonstrated that oxygen concentration and nitrification had similar patterns suggesting that nitrification may be limited by oxygen penetration into sediments. We conclude that temperature and sediment NH₄ can be useful for predicting broad-scale temporal and spatial nitrification patterns, respectively, but oxygen penetration into the sediments likely regulates nitrification rates in much of the UMR. Overall, we estimated that nitrification produces 6982 mt N/y of NO₃⁻ or 7% of the total annual NO₃⁻ budget.