Flux-gradient relationships based upon similarity theory have been reported to severely underestimate scalar fluxes in the roughness sublayer above forests, as compared to independent flux estimates (for example, eddy covariance or energy balance measurements). This paper presents the results of a unique three-month investigation into the validity of similarity theory in the roughness sublayer above forests. Eddy covariance and flux-gradient measurements of carbon dioxide (CO2) exchange were compared above a mixed deciduous forest at Camp Borden, Ontario, both before and after leaf senescence. The eddy covariance measurements used a Li-Cor infrared gas analyzer, and the flux-gradient (similarity theory) measurements featured a tunable diode laser Trace Gas Analysis System (TGAS). The TGAS resolved the CO2 concentration difference to 300 parts per trillion by volume (ppt) based upon a half-hour sampling period. The measured enhancement factor γ (the ratio of independent flux estimates, in this case eddy covariance, to similarity theory fluxes) was smaller and occurred closer to the canopy than in most previous investigations of similarity theory. Very good agreement between the eddy covariance and similarity theory fluxes was found between 1.9 and 2.2 canopy heights (hc), and the mean enhancement factors measured before and after leaf senescence were 1.10 ± 0.06 and 1.24 ± 0.07, respectively. Larger discrepancies were measured closer to the canopy (1.2 to 1.4 hc), and mean enhancement factors of 1.60 ± 0.10 and 1.82 ± 0.11 were measured before and after leaf senescence, respectively. Overall, the Borden results suggest that similarity theory can be used within the roughness sublayer with a greater confidence than previously has been believed.