We measured the stable isotope ratio of respired carbon dioxide at two spatial scales in a black spruce forest in northern Canada: CO2 released from the forest floor and CO2 released from the entire ecosystem at night. Despite wide variation in the δ13C values of organic matter among above-ground plant species, and along a continuum from moss through to the mineral soil, the carbon isotope ratio of respired CO2 was quite similar to the δ13C value for the dominant black spruce foliage. The CO2 released from the forest floor during the fall was slightly enriched in 13O compared to CO2 respired by the entire ecosystem, perhaps because soil respiration contributes a larger percentage to total ecosystem respiration later in the year as the soil warms. Short-term changes in the oxygen isotope ratio of precipitation and variation in enrichment of 18O during evaporation and transpiration had significant effects on the δ18O value of respired CO2. Changes in the oxygen isotope ratio of water in moss tissue can have a large effect on total ecosystem respired CO2 both because a large surface area is covered by moss tissue in this ecosystem and because the equilibration between CO2 diffusing through the moss and water in moss tissue is very rapid. During the summer we observed that the δ18O value of CO2 respired from the forest floor was relatively depleted in 18O compared to CO2 respired from the entire ecosystem. This was because water in black spruce foliage had higher δ18O values than moss and soil water, even at night when transpiration had stopped.