Vertical transport of surface emission to the free troposphere, usually associated with frontal lifting in warm conveyor belts or ascent in deep convection, has significant influence on the vertical structure of atmospheric trace gases. Consequently, it may impact estimates of the surface fluxes of these gases inferred from remote sensing observations that are based on thermal infrared radiances (TIR), since these measurements are sensitive mainly to signals in the free troposphere. In this work, we assessed the sensitivity of regional CO source estimates to the vertical CO distribution, by assimilating multi-spectral MOPITT V5J CO retrievals with the GEOS-Chem model. We compared the source estimates obtained by assimilating the CO profiles and the surface layer retrievals from June 2004 to May 2005. The inversion analyses all produced a reduction in CO emissions in the tropics and subtropics and an increase in the extratropics. The tropical decreases were particularly pronounced for regions where the biogenic source of CO was dominant, suggesting an overestimate of the a priori isoprene source of CO in the model. We found that the differences between the regional source estimates inferred from the profile and surface layer retrievals for 2004–2005 were small, generally less than 5% for the main continental regions, except for estimates for South Asia, North America, and Europe. Because of discrepancies in convective transport in the model, the CO source estimates for India and Southeast Asia inferred from the CO profiles were significantly higher than those estimated from the surface layer retrievals during June–August 2004. On the other hand, the profile inversion underestimated the CO emissions from North America and Europe compared to the assimilation of the surface layer retrievals. We showed that vertical transport of air from the North American and European boundary layer is slower than from other continental regions and thus air in the free troposphere from North America and Europe is more chemically aged, which could explain the discrepancy between the source estimates inferred from the profile and surface layer retrievals. We also examined the impact of the OH distribution on the source estimates using OH fields from versions v5-07-08 and v8-02-01 of GEOS-Chem. The impact of the different OH fields was particularly large for the extratropical source estimates. For example, for North America, using the surface layer retrievals, we estimated a total CO source of 37 and 55 Tg CO with the v5-07-08 and v8-02-01 OH fields, respectively, for June–August 2004. For Europe the source estimates were 57 and 72 Tg CO, respectively. We found that the discrepancies between the source estimates obtained with the two OH fields were larger when using the profile data, which is consistent with greater sensitivity to the more chemically aged air in the free troposphere. Our findings indicate that regional CO source estimates are sensitive to the vertical CO structure. They suggest that assimilating a broader range of composition measurements to provide better constraint on tropospheric OH and the biogenic sources of CO is essential for reliable quantification of the regional CO budget.