Growing interest in the potential for agricultural soils to provide a sink for atmospheric C has prompted studies of effects of management on soil organic carbon (SOC) sequestration. We analyzed the impact on SOC of four N fertilization rates (0–270 kg N ha⁻¹) and four cropping systems: continuous corn (CC) (Zea mays L.); corn–soybean [Glycine max (L.) Merr.] (CS); corn–corn–oat–alfalfa (oat, Avena sativa L.; alfalfa, Medicago sativa L.) (CCOA), and corn–oat–alfalfa–alfalfa (COAA). Soils were sampled in 2002, Years 23 and 48 of the experiments located in northeast and north-central Iowa, respectively. The experiments were conducted using a replicated split-plot design under conventional tillage. A native prairie was sampled to provide a reference (for one site only). Cropping systems that contained alfalfa had the highest SOC stocks, whereas the CS system generally had the lowest SOC stocks. Concentrations of SOC increased significantly between 1990 and 2002 in only two of the nine systems for which historical data were available, the fertilized CC and COAA systems at one site. Soil quality indices such as particulate organic carbon (POC) were influenced by cropping system, with CS < CC < CCOA. In the native prairie, SOC, POC, and resistant C concentrations were 2.8, 2.6, and 3.9 times, respectively, the highest values in cropped soil, indicating that cultivated soils had not recovered to precultivation conditions. Although corn yields increased with N additions, N fertilization increased SOC stocks only in the CC system at one site. Considering the C cost for N fertilizer production, N fertilization generally had a net negative effect on C sequestration.