The major driving factors of soil CO2 production – substrate supply, temperature, and water content – vary vertically within the soil profile, with the greatest temporal variations of these factors usually near the soil surface. Several studies have demonstrated that wetting and drying of the organic horizon contributes to temporal variation in summertime soil CO2 efflux in forests, but this contribution is difficult to quantify. The objectives of this study were to partition CO2 production vertically in a mixed hardwood stand of the Harvard Forest, Massachusetts, USA, and then to use that partitioning to evaluate how the relative contributions of CO2 production by genetic soil horizon vary seasonally and interannually. We measured surface CO2 efflux and vertical soil profiles of CO2 concentration, temperature, water content, and soil physical characteristics. These data were applied to a model of effective diffusivity to estimate CO2 flux at the top of each genetic soil horizon and the production within each horizon. A sensitivity analysis revealed sources of uncertainty when applying a diffusivity model to a rocky soil with large spatial heterogeneity, especially estimates of bulk density and volumetric water content and matching measurements of profiles and surface fluxes. We conservatively estimate that the O horizon contributed 40–48% of the total annual soil CO2 efflux. Although the temperature sensitivity of CO2 production varied across soil horizons, the partitioning of CO2 production by horizon did not improve the overall prediction of surface CO2 effluxes based on temperature functions. However, vertical partitioning revealed that water content covaried with CO2 production only in the O horizon. Large interannual variations in estimates of O horizon CO2 production indicate that this layer could be an important transient interannual source or sink of ecosystem C.