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Abstract The carbon dioxide (CO2) fluxes from headwater streams are not well quantified and could be a source of significant carbon, particularly in systems underlain by carbonate lithology. Also, the sensitivity of carbonate systems to changes in temperature will make these fluxes even more significant as climate changes. This study quantifies small-scale CO2 efflux and estimates annual CO2 emission from a headwater stream at the Konza Prairie Long-Term Ecological Research Site and Biological Station (Konza), in a complex terrain of horizontal, alternating limestones and shales with small-scale karst features. CO2 effluxes ranged from 2.2 to 214 g CO2 m−2 day−1 (mean: 20.9 CO2 m−2 day−1). Downstream of point groundwater discharge sources, CO2 efflux decreased, over 2 m, to 3–40% of the point-source flux, while δ13C-CO2 increased, ranging from −9.8 ‰ to −23.2 ‰ V-PDB. The δ13C-CO2 increase was not strictly proportional to the CO2 flux but related to the origin of vadose zone CO2. The high spatial and temporal variability of CO2 efflux from this headwater stream informs those doing similar measurements and those working on upscaling stream data, that local variability should be assessed to estimate the impact of headwater stream CO2 efflux on the global carbon cycle.