Theoretical models predict a marked increase in atmospheric O 2 to ∼35% during the Permo-Carboniferous (∼300 Ma) occurring against a low (∼0.03%) CO 2 level. An upper O 2 value of 35%, however, remains disputed because ignition data indicate that excessive global forest fires would have ensued. This uncertainty limits interpretation of the role played by atmospheric oxygen in Late Paleozoic biotic evolution. Here, we describe new results from laboratory experiments with vascular land plants that establish that a rise in O 2 to 35% increases isotopic fractionation (Δ 13 C) during growth relative to control plants grown at 21% O 2 . Despite some effect of the background atmospheric CO 2 level on the magnitude of the increase, we hypothesize that a substantial Permo-Carboniferous rise in O 2 could have imprinted a detectable geochemical signature in the plant fossil record. Over 50 carbon isotope measurements on intact carbon from four fossil plant clades with differing physiological ecologies and ranging in age from Devonian to Cretaceous reveal a substantial Δ 13 C anomaly (5‰) occurring between 300 and 250 Ma. The timing and direction of the Δ 13 C excursion is consistent with the effects of a high O 2 atmosphere on plants, as predicted from photosynthetic theory and observed in our experiments. Preliminary calibration of the fossil Δ 13 C record against experimental data yields a predicted O 2 /CO 2 mixing ratio of the ancient atmosphere consistent with that calculated from long-term models of the global carbon and oxygen cycles. We conclude that further work on the effects of O 2 in the combustion of plant materials and the spread of wildfire is necessary before existing data can be used to reliably set the upper limit for paleo-O 2 levels.