The stable carbon isotopic compositions of alkenones have been used to interpret the long-term history of the partial pressure of atmospheric carbon dioxide (pCO2). Although extensive water column and culture studies document the potential utility and limitations of this approach, to date the accuracy of pCO2 values derived from sedimentary alkenones remains untested. For this study we establish Holocene-aged, alkenone-based CO2aq estimates (2aq>alk) from 20 sites along a central Pacific Ocean transect and compare them against both observed modern water column CO2aq and estimated preindustrial concentrations at the depth of alkenone production at each site. Although the 2aq>alk track measured water column values, they are conspicuously lower than modern values across the subtropics. This offset likely reflects the contributions of anthropogenic CO2 in modern surface waters relative to preindustrial concentrations at the time of alkenone production. When a model-based estimate of anthropogenic CO2 is removed from the modern observed values, a majority (84%) of 2aq>alk falls within 20% of modeled preindustrial values. Consistency between the modeled and alkenone-based estimates of preindustrial CO2 levels points to the relative accuracy of the alkenone-CO2 method across a wide range of ocean and biogeographic regimes, provided that phosphate concentrations, at the depth of haptophyte production, are reasonably constrained. It further suggests that light-limited growth and/or active carbon uptake, if they occur, have a negligible effect on reconstructed 2aq>.