Separating ecosystem and soil respiration into autotrophic and heterotrophic component sources is necessary for understanding how the net ecosystem exchange of carbon (C) will respond to current and future changes in climate and vegetation. Here, we use an isotope mass balance method based on radiocarbon to partition respiration sources in three mature black spruce forest stands in Alaska. Radiocarbon (Δ14C) signatures of respired C reflect the age of substrate C and can be used to differentiate source pools within ecosystems. Recently-fixed C that fuels plant or microbial metabolism has Δ14C values close to that of current atmospheric CO2, while C respired from litter and soil organic matter decomposition will reflect the longer residence time of C in plant and soil C pools. Contrary to our expectations, the Δ14C of C respired by recently excised black spruce roots averaged 14‰ greater than expected for recently fixed photosynthetic products, indicating that some portion of the C fueling root metabolism was derived from C storage pools with turnover times of at least several years. The Δ14C values of C respired by heterotrophs in laboratory incubations of soil organic matter averaged 60‰ higher than the contemporary atmosphere Δ14CO2, indicating that the major contributors to decomposition are derived from a combination of sources consistent with a mean residence time of up to a decade. Comparing autotrophic and heterotrophic Δ14C end members with measurements of the Δ14C of total soil respiration, we calculated that 47–63% of soil CO2 emissions were derived from heterotrophic respiration across all three sites. Our limited temporal sampling also observed no significant differences in the partitioning of soil respiration in the early season compared with the late season. Future work is needed to address the reasons for high Δ14C values in root respiration and issues of whether this method fully captures the contribution of rhizosphere respiration.