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Urban areas are directly or indirectly responsible for the majority of anthropogenic CO2 emissions. In this study, we characterize observed atmospheric CO2 mixing ratios and estimated CO2 fluxes at three sites across an urban-to-rural gradient in Boston, MA, USA. CO2 is a well-mixed greenhouse gas, but we found significant differences across this gradient in how, where, and when it was exchanged. Total anthropogenic emissions were estimated from an emissions inventory and ranged from 1.5 to 37.3 mg·C·ha−1·yr−1 between rural Harvard Forest and urban Boston. Despite this large increase in anthropogenic emissions, the mean annual difference in atmospheric CO2 between sites was approximately 5% (20.6 ± 0.4 ppm). The influence of vegetation was also visible across the gradient. Green-up occurred near day of year 126, 136, and 141 in Boston, Worcester and Harvard Forest, respectively, highlighting differences in growing season length. In Boston, gross primary production—estimated by scaling productivity by canopy cover—was ~75% lower than at Harvard Forest, yet still constituted a significant local flux of 3.8 mg·C·ha−1·yr−1. In order to reduce greenhouse gas emissions, we must improve our understanding of the space-time variations and underlying drivers of urban carbon fluxes.