Accurate estimates of root respiration are crucial to predicting belowground C cycling in forest ecosystems. Inhibition of respiration has been reported as a short-term response of plant tissue to elevated measurement [CO2]. We sought to determine if measurement [CO2] affected root respiration in samples from mature sugar maple (Acer saccharum Marsh.) forests and to assess possible errors associated with root respiration measurements made at [CO2]s lower than that typical of the soil atmosphere. Root respiration was measured as both CO2 production and O2 consumption on excised fine roots (≤ 1.0 mm) at [CO2]s ranging from 350 to > 20,000 μl l−1. Root respiration was significantly affected by the [CO2] at which measurements were made for both CO2 production and O2 consumption. Root respiration was most sensitive to [CO2] near and below normal soil concentrations (< 1500 μl l−1). Respiration rates changed little at [CO2]s above 3000 μl l−1 and were essentially constant above 6000 μl l−1 CO2. These findings call into question estimates of root respiration made at or near atmospheric [CO2], suggesting that they overestimate actual rates in the soil. Our results indicate that sugar maple root respiration at atmospheric [CO2] (350 μl l−1) is about 139% of that at soil [CO2]. Although the causal mechanism remains unknown, the increase in root respiration at low measurement [CO2] is significant and should be accounted for when estimating or modeling root respiration. Until the direct effect of [CO2] on root respiration is fully understood, we recommend making measurements at a [CO2] representative of, or higher than, soil [CO2]. In all cases, the [CO2] at which measurements are made and the [CO2] typical of the soil atmosphere should be reported.