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Soil-test biological activity (STBA) is a key indicator describing the functions of soil to catabolize organic amendments, promote soil organic C sequestration, and cycle nutrients. We explored how climatic zone, land use, and physiographic region affected the relationship of STBA with several soil fertility properties and processes. Soils from Georgia, North Carolina, Pennsylvania, and Virginia (wet climate) were Aquults, Fluvents, Udalfs, Udepts, and Udults and soils from Nebraska and Oklahoma (dry climate) were Udolls and Ustolls. Annual cropland and perennial pasture were the land uses. The Prairie region had silt loam and silty clay loams, the Ridge–Valley region had silt loams, and the Piedmont and Coastal Plain regions had fine sandy loam, loam, sandy clay loam, and sandy loams. Across all locations, STBA with the flush of CO2 during 3 d following rewetting of dried soil was most strongly associated with cumulative C mineralization, basal soil respiration, particulate organic C, potential N mineralization during 24 d of aerobic incubation, residual soil ammonium, soil microbial biomass C, and total soil N. Climatic zone modified the association between STBA and net N mineralization, possibly caused by greater N immobilization in drier soils with 2:1 clays. Physiographic region had a small effect on the relationship between STBA and net N mineralization. Land use did not alter this relationship significantly. Soil-test biological activity was verified as a robust indicator of soil organic N supply, as well as of general soil biological condition to assess the soil’s catabolic potential and potential C storage.