The current emphasis on global climate studies has led the scientific community to set up a number of sites for measuring the long-term biosphere-atmosphere net CO₂ exchange (net ecosystem exchange, NEE). Partitioning this flux into its elementary components, net assimilation (F _A ), and respiration (F _R ), remains necessary in order to get a better understanding of biosphere functioning and design better surface exchange models. Noting that F _R and F _A have different isotopic signatures, we evaluate the potential of isotopic ¹³CO₂ measurements in the air (combined with CO₂ flux and concentration measurements) to partition NEE into F _R and F _A on a routine basis. The study is conducted at a temperate coniferous forest where intensive isotopic measurements in air, soil, and biomass were performed in summer 1997. The multilayer soil-vegetation-atmosphere transfer model MuSICA is adapted to compute ¹³CO₂ flux and concentration profiles. Using MuSICA as a “perfect” simulator and taking advantage of the very dense spatiotemporal resolution of the isotopic data set (341 flasks over a 24-hour period) enable us to test each hypothesis and estimate the performance of the method. The partitioning works better in midafternoon when isotopic disequilibrium is strong. With only 15 flasks, i.e., two ¹³CO₂ nighttime profiles (to estimate the isotopic signature of F _R ) and five daytime measurements (to perform the partitioning) we get mean daily estimates of F _R and F _A that agree with the model within 15–20%. However, knowledge of the mesophyll conductance seems crucial and may be a limitation to the method.