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Although C₄ plant expansions have been recognized in the late Miocene, identification of the underlying causes is complicated by the uncertainties associated with estimates of ancient precipitation, temperature, and partial pressure of atmospheric carbon dioxide (P co ₂). Here we report the carbon isotopic compositions of leaf waxn-alkanes in lake sediment cores from two sites in Mesoamerica that have experienced contrasting moisture variations since the last glacial maximum. Opposite isotopic trends obtained from these two sites indicate that regional climate exerts a strong control on the relative abundance of C₃ and C₄ plants and that in the absence of favorable moisture and temperature conditions, lowP co ₂ alone is insufficient to drive an expansion of C₄ plants.