Abstract. We have developed an ensemble Kalman Filter (EnKF) to estimate 8-day regional surface fluxes of CO₂ from space-borne CO₂ dry-air mole fraction observations (X_CO2) and evaluate the approach using a series of synthetic experiments, in preparation for data from the NASA Orbiting Carbon Observatory (OCO). The 32-day duty cycle of OCO alternates every 16 days between nadir and glint measurements of backscattered solar radiation at short-wave infrared wavelengths. The EnKF uses an ensemble of states to represent the error covariances to estimate 8-day CO₂ surface fluxes over 144 geographical regions. We use a 12×8-day lag window, recognising that X_CO2 measurements include surface flux information from prior time windows. The observation operator that relates surface CO₂ fluxes to atmospheric distributions of X_CO2 includes: a) the GEOS-Chem transport model that relates surface fluxes to global 3-D distributions of CO₂ concentrations, which are sampled at the time and location of OCO measurements that are cloud-free and have aerosol optical depths <0.3; and b) scene-dependent averaging kernels that relate the CO₂ profiles to X_CO2, accounting for differences between nadir and glint measurements, and the associated scene-dependent observation errors. We show that OCO X_CO2 measurements significantly reduce the uncertainties of surface CO₂ flux estimates. Glint measurements are generally better at constraining ocean CO₂ flux estimates. Nadir X_CO2 measurements over the terrestrial tropics are sparse throughout the year because of either clouds or smoke. Glint measurements provide the most effective constraint for estimating tropical terrestrial CO₂ fluxes by accurately sampling fresh continental outflow over neighbouring oceans. We also present results from sensitivity experiments that investigate how flux estimates change with 1) bias and unbiased errors, 2) alternative duty cycles, 3) measurement density and correlations, 4) the spatial resolution of estimated flux estimates, and 5) reducing the length of the lag window and the size of the ensemble. At the revision stage of this manuscript, the OCO instrument failed to reach its orbit after it was launched on 24 February 2009. The EnKF formulation presented here is also applicable to GOSAT measurements of CO₂ and CH₄.