Carbon dioxide respiration rates were measured in the laboratory at −2°C for 88 arctic Alaska soil horizon samples. Soil horizon samples represented most ATLAS C-flux study sites to a 1-m depth. Sites represented extend across the Alaska Arctic, from tundra at Prudhoe Bay (70°N, 149°W) in the northeast to the tundra–forest transition zone of the Seward Peninsula (64°N, 163°W) in the southwest, a range of nearly 1000 km. Results of an initial temperature sensitivity study indicated that at 4°C, organic horizons (>20% organic carbon [OC]) have average respiration rates that are twice as high as those found for the mineral horizons. However, as temperature dropped to −2°C, respiration in organic horizons was about the same as that for the low OC mineral horizons (with <5% OC). However, organic enriched mineral horizons (with OC 5–20%) maintained higher rates of respiration at the subzero temperature, and rates averaged 1.7 times higher than for the organics at −2°C. Results of −2°C incubations for all study samples indicated that, indeed, there is a good correlation between soil water-soluble OC (wsOC) stocks and CO2 respiration rates in both organic surface horizons and among the high respiring organic enriched mineral horizons, R2 = 0.70 (p < 0.01) and R2 = 0.62 (p < 0.01), respectively. The presence of permafrost was associated with increased wsOC substrate levels, and the highest respiration rate found over all samples was in an organic enriched (17.8% OC) mineral horizon from the permafrost layer. High levels of substrates and high respiration rates were associated with surface organic horizons from sites at the forest–tundra transition (Council) for all sites with shrub vegetation and for Eriophorum tussocks at Toolik Lake. Stocks of wsOC were not correlated to soil total OC levels. These results point to the importance of a soil carbon–substrate shift with subzero temperature and its implications for cold-season processes throughout the soil active layer.