Though the positive response of soil organic matter decay rates to temperature is theorized to decline with the bioreactivity (carbon normalized respiration) of organic matter, studies only sometimes support this idea. One potential reason for discrepancies among studies is the isolation of soil horizons in incubation experiments, which may limit the exchange of substrates among horizons that occurs in situ and in incubations employing intact, multiple-horizon cores, and thus may limit stimulation of microbial activities. To what degree does the isolation of individual soil horizons influence our ability to predict temperature sensitivity of respiration? Addressing this question is important, because incubation studies are frequently used to parameterize ecosystem process models and to formulate at least qualitative predictions of potential SOC destabilization in future climate scenarios.To address this question, we conducted three parallel incubation experiments using soil collected from podzolic boreal forest sites in two regions similar in vegetation and soil type, but that differ in climate. The experiments consisted of (1) intact unaltered L, F, H horizons as a whole unit (hereafter called LFH), (2) isolated horizons from the same LFH, and (3) rebuilt LFH of those isolated horizons. The soils were incubated at 5 °C, 10 °C, and 15 °C with greater than 430 days of incubation with soil respiration measured at 6 time points.Cumulative respiration was greater in the soils collected from the higher latitude region (hereafter cold region) relative to those collected from the lower latitude region (hereafter warm region) regardless of incubation temperature or experiment, suggesting that the warm region soils are less bioavailable. The temperature sensitivity (Q10) of soil respiration, however, was influenced by whether the organic horizons were intact, isolated, or rebuilt. Respiratory responses of the LFH computed from the sum of isolated horizons were not different between the two regions (Q10 of 2.84 ± 0.10 and 2.72 ± 0.07 for cold and warm regions, respectively). In contrast, the respiratory responses of the more realistic rebuilt LFH over the entire experiment were significantly higher, and different between regions (3.52 ± 0.12 and 4.68 ± 0.16 for cold and warm regions, respectively). These results are congruent with trends observed in the intact unaltered LFH, and speak to the likely importance of substrate exchange among soil horizons as a driver of aggregated respiratory responses to temperature. The flow of labile substrates across or among horizons may facilitate the decomposition of relatively complex substrates exhibiting higher activation energy of decay. This exchange of labile substrates could promote relatively greater temperature responses of soil respiratory CO2 losses. Based on these results, we suggest that a full understanding of the temperature sensitivity of SOC transformations requires using soil samples that encompass multiple soil horizons.