Nickel is a trace metal that exhibits pronounced long-term immobilization reactions in soil. It is unknown if the slowly decreasing solubility of Ni in soil on aging correlates with decreased toxicity to soil biota. Three uncontaminated soils (pH 4.5–7.6) were contaminated with NiCl2 and experimentally leached or incubated outdoors with free drainage for up to 15 months. Nickel toxicity was measured for three microbial processes (potential nitrification rate, glucose-induced respiration, and C mineralization of maize residue). Results for leached and aged samples were compared with results for these soils tested immediately after spiking. Experimental leaching increased Ni ED50s (Ni dose to inhibit process by 50%) with a median factor of 2.0, whereas Ni ED50s in soils aged 15 months were a factor 1 to 23 (median, 4.6) larger compared to freshly spiked soils. Changes in soil Ni toxicity on aging generally were largest in the soil with the highest pH, consistent with the largest relative decreases of soil solution Ni concentration or predicted Ni2+ activity. Soil solution Ni concentrations explained part, but not all, of the reduction in Ni toxicity. The predicted soil solution Ni2+ activity also did not fully explain the reduced toxicity, which was ascribed to the variable concentrations of ions competing with Ni2+ at biological membranes (e.g., H+, Mg2+, or Ca2+) among treatments. It is concluded that testing Ni toxicity to soil microbial processes immediately after spiking soils in the laboratory overestimates Ni toxicity compared to aged soils. Soil solution composition in freshly spiked soils clearly is different from that in leached or aged soils; therefore, soil spiked with metal salts should be leached before toxicity tests begin.