An estimate of soil mineralizable N is needed to determine crop needs for N fertilizer. The objective of this research was to estimate soil net N mineralization in soils maintained in continuous corn (Zea mays L.) (CC), corn-soybean [Glycine max (L.) Merr.] (CS), and corn-soybean-wheat (Triticum aestivum L.)/alfalfa (Medicago sativa L.)-alfalfa (CSWA) rotations that have been managed since 1990 with zero N (0N), low N (LN), and high N (HN) fertilization. Soil samples were taken from 0- to 20-cm depth in plots planted to corn in 1998. In order to produce more realistic time-series data of net N mineralization, soils were incubated in filtration units in a variable-temperature incubator (VTI) that mimicked field soil temperatures under a growing corn canopy. Rotation and N fertilization significantly affected net N mineralization in soil samples. Cumulative net N mineralized in a 189-d field temperature incubation averaged 133 +/- 6 kg ha(-1) in CC, 142 +/- 5 kg ha(-1) in CS, and 189 +/- 5 kg ha(-1) in CSWA. Across rotations, average net N mineralized was 166 +/- 9 kg ha(-1) in ON plots, 147 +/- 10 kg ha(-1) in LN plots, and 152 +/- 10 kg ha(-1) in HN plots. Inclusion of a legume, particularly alfalfa, in the rotation increased net N mineralized. Generally, more net N was mineralized from plots receiving no fertilizer N than from soil with a history of N fertilization. Variable-temperature incubation produced realistic time series data with low sample variability.