The alkenone–
p
CO
2
methodology has been used to reconstruct the partial pressure of ancient atmospheric carbon dioxide (
p
CO
2
) for the past 45 million years of Earth's history (Middle Eocene to Pleistocene epochs). The present long-term CO
2
record is a composite of data from multiple ocean localities that express a wide range of oceanographic and algal growth conditions that potentially bias CO
2
results. In this study, we present a
p
CO
2
record spanning the past 40 million years from a single marine locality, Ocean Drilling Program Site 925 located in the western equatorial Atlantic Ocean. The trends and absolute values of our new CO
2
record site are broadly consistent with previously published multi-site alkenone–CO
2
results. However, new
p
CO
2
estimates for the Middle Miocene are notably higher than published records, with average
p
CO
2
concentrations in the range of 400–500 ppm. Our results are generally consistent with recent
p
CO
2
estimates based on boron isotope-pH data and stomatal index records, and suggest that CO
2
levels were highest during a period of global warmth associated with the Middle Miocene Climatic Optimum (17–14 million years ago, Ma), followed by a decline in CO
2
during the Middle Miocene Climate Transition (approx. 14 Ma). Several relationships remain contrary to expectations. For example, benthic foraminiferal
δ
18
O records suggest a period of deglaciation and/or high-latitude warming during the latest Oligocene (27–23 Ma) that, based on our results, occurred concurrently with a long-term decrease in CO
2
levels. Additionally, a large positive
δ
18
O excursion near the Oligocene–Miocene boundary (the Mi-1 event, approx. 23 Ma), assumed to represent a period of glacial advance and retreat on Antarctica, is difficult to explain by our CO
2
record alone given what is known of Antarctic ice sheet history and the strong hysteresis of the East Antarctic Ice Sheet once it has grown to continental dimensions. We also demonstrate that in the Neogene with low CO
2
levels, algal carbon concentrating mechanisms and spontaneous biocarbonate–CO
2
conversions are likely to play a more important role in algal carbon fixation, which provides a potential bias to the alkenone–
p
CO
2
method.
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