We explore seasonal variability in isotopic fractionation by analyzing observational data from the atmosphere and the biosphere, as well as simulated data from a global model. Using simulated values of atmospheric CO2 and its carbon isotopic composition, we evaluated different methods for specifying background concentrations when calculating the isotopic signature of source CO2 (δs) to the atmosphere. Based on this evaluation, we determined that free troposphere measurements should be used when available as a background reference when calculating δs from boundary layer observations. We then estimate the seasonal distribution of δs from monthly climatologies derived from several atmospheric sampling sites. This approach yields significant seasonal variations in δs with more enriched values during the summer months that exceed the uncertainty of δs estimated for any given month. Intra-annual measurements of δ13C in the cellulose of Pinus taeda growing in the southeastern U.S. also reveal seasonal isotopic variations that are consistent in phase but not necessarily amplitude with atmospherically derived estimates of δs. Coherent seasonal patterns in δs inferred from the atmosphere and observed in the biosphere were not consistent with the seasonal isotopic discrimination simulated by a commonly used biosphere model. However, δs seasonality consistent with observations from the atmosphere and biosphere was retrieved with a revised biosphere model when stomatal conductance, and thus isotopic discrimination, was allowed to vary in response to vapor pressure deficit rather than relative humidity. Therefore, in regions where vapor pressure deficit and relative humidity are positively covariant over the growth season, such as the sub-tropics, different stomatal conductance models may yield very different estimates of CO2 and H2O exchange between the biosphere and atmosphere.