Our objective was to measure the carbon isotope ratio of CO2 released by respiration (δr) within forest canopies at different times during the growing season and to use this information to estimate forest ecosystem carbon isotope discrimination. We made measurements in the three major forest types (black spruce, jack pine, and aspen) at the southern and northern ends of the boreal forest in central Canada. This research was part of a larger study, the Boreal Ecosystem-Atmosphere Study (BOREAS). The δr values, calculated from measurements of change in the concentration and carbon isotope ratio of atmospheric CO2 in air samples collected at night, ranged from −28.1‰ to −25.9‰ with an average (± s.d.) of −26.‰ ± 0.5‰. There was good correlation between calculated δr values and measurements of (1) the carbon isotope ratio of CO2 released directly from the soil and (2) the δ13C values of foliage collected from the dominant tree species at each site. Carbon isotope discrimination during photosynthetic gas exchange (ΔA) by each forest ecosystem was estimated as the difference between the carbon isotope ratio of atmospheric CO2 at the top of the canopy (δa) and the isotopic composition of respired CO2: ΔA = δa − δr. All three of the major forest types had similar values of ΔA, with an average (± s.d.) of 19.1‰ ± 0.5‰. However, a seasonal change in forest discrimination was observed for aspen forests in both the northern and southern study areas, with an increase in ΔA occurring between the middle and end of the growing season. In contrast, the evergreen conifer canopies exhibited relatively constant discrimination values throughout the active growing season.