AbstractThe Atmospheric Carbon Transport (ACT)‐America Earth Venture mission conducted five airborne campaigns across four seasons from 2016 to 2019, to study the transport and fluxes of Greenhouse gases across the eastern United States. Unprecedented spatial sampling of atmospheric tracers (CO₂, carbon monoxide [CO], carbonyl sulfide [COS]) related to biospheric processes offers opportunities to improve our qualitative and quantitative understanding of seasonal and spatial patterns of biospheric carbon uptake. Here, we examine co‐variation of boundary layer enhancements of CO₂, CO, and COS across three diverse regions: the crop‐dominated Midwest, evergreen‐dominated South, and deciduous broadleaf‐dominated Northeast. To understand the biogeochemical processes controlling these tracers, we compare the observed co‐variation to simulated co‐variation resulting from model‐ and satellite‐ constrained surface carbon fluxes. We found indication of a common terrestrial biogenic sink of CO₂ and COS and secondary production of CO from biogenic sources in summer throughout the eastern US, driven by stomatal conductance. Upper Midwest crops drive CO₂ and COS depletion from early to late summer. Northeastern temperate forests drive CO₂ and COS depletion in late summer. The unprecedented ACT‐America flask samples uncovered evidence that southern humid temperate forests photosynthesize and absorb CO₂ and COS, and emit CO precursors, deep into the growing season. Satellite‐ constrained carbon fluxes capture much of the observed seasonal and spatial variability, but underestimate the magnitude of net CO₂ and COS depletion in the South, indicating a stronger than expected net sink of CO₂ in late summer. Additional sampling of the South will more accurately constrain underlying biological processes and climate sensitivities governing southern carbon dynamics.