Temperate forests play an important role in the global carbon cycle, and are thought to currently be a sink for atmospheric CO2. However, we lack understanding of the drivers of forest carbon accumulation and loss, hampering our ability to predict carbon cycle responses to global change. In this study, we used CO2 flux and radiocarbon (14C) measurements to investigate the role of seasonal drivers on soil respiration. Radiocarbon measurements of CO2 evolved during incubation of fine roots and root-free soils at the beginning and end of the growing season (April and August) showed that these two soil respiration sources (fine roots vis-à-vis soils) have different mean residence times that stayed constant between seasons. Radiocarbon measurements show that root respiration was made up of carbon fixed 3–5 years prior to sampling, and that heterotrophic respiration was made up of carbon fixed 7–10 years prior. The difference in radiocarbon signature between the two sources allowed us to partition autotrophic and heterotrophic respiration sources for soil respiration measurements in the field. We observed a small but significant increase in ∆14C of soil respiration between April and August, suggesting an increase in heterotrophic respiration sources over the growing season. Using a two end-member mixing model, we estimate that 55 ± 22% of soil respiration originated from autotrophic (root) sources in April, but their contribution dropped to 38 ± 21% in August. These findings suggest that the contribution of root respiration increases at a time of high productivity and/or as a result of relatively low microbial respiration in the early spring in this old-growth coniferous forest.