High grain production of corn (Zea mays L.) can be maintained by adding inorganic N fertilizer, and also by using crop rotations that include alfalfa (Medicago sativa L.), but the relative impact of these management practices on soil quality is uncertain. We examined the effects on soil of N fertilization rate (0, 90, 180, 270 kg ha⁻¹, corn phase only) in four cropping systems: CC, continuous corn; CS, corn–soybean [Glycine max (L.) Merr.]; CCOA, corn–corn–oat (Avena sativa L.)–alfalfa; and corn–oat–alfalfa–alfalfa (COAA). The 23- and 48-yr-old experimental sites, situated in northeast (Nashua) and north central (Kanawha) Iowa, were in a replicated split-plot design and managed with conventional tillage. At Nashua, we measured available N, potential net N mineralization and microbial biomass C (MBC) throughout the growing season; all were significantly higher in the CCOA system. At both sites, post-harvest N stocks, and soil organic C (SOC) concentrations were significantly higher in systems containing alfalfa. Grain yield was most strongly correlated with soil N properties. At Nashua, N fertilizer additions resulted in significantly lower soil pH (0- to 15-cm depth) and lower exchangeable Ca, Mg, and K and cation exchange capacity (CEC) in the CC and CCOA systems. In an undisturbed prairie reference site for Nashua, low available N, low pH, and high CEC suggested a strong influence of the vegetation on nutrient cycling. In terms of management of soil fertility, inclusion of alfalfa in the rotation differed fundamentally from addition of N fertilizer because high yield was maintained with fewer adverse effects on soil quality.