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Net photosynthesis is the largest single flux in the global carbon cycle, but controls over its variability are poorly understood because there is no direct way of measuring it at the ecosystem scale. We report observations of ecosystem carbonyl sulfide (COS) and CO2 fluxes that resolve key gaps in an emerging framework for using concurrent COS and CO2 measurements to quantify terrestrial gross primary productivity. At a wheat field in Oklahoma we found that in the peak growing season the flux-weighted leaf relative uptake of COS and CO2 during photosynthesis was 1.3, at the lower end of values from laboratory studies, and varied systematically with light. Due to nocturnal stomatal conductance, COS uptake by vegetation continued at night, contributing a large fraction (29%) of daily net ecosystem COS fluxes. In comparison, the contribution of soil fluxes was small (1-6%) during the peak growing season. Upland soils are usually considered sinks of COS. In contrast, the well-aerated soil at the site switched from COS uptake to emissions at a soil temperature of around 15 °C. We observed COS production from the roots of wheat and other species and COS uptake by root-free soil up to a soil temperature of around 25 °C. Our dataset demonstrates that vegetation uptake is the dominant ecosystem COS flux in the peak growing season, providing support of COS as an independent tracer of terrestrial photosynthesis. However, the observation that ecosystems may become a COS source at high temperature needs to be considered in global modeling studies.