The oxygen isotope ratio of CO2 liberated from structural carbonate in tooth enamel apatite was measured at phosphoric acid reaction temperatures of 25°C, 60°C and 90°C, and it was found that apparent acid fractionation factors for pristine enamel, fossilized enamel, and calcite follow different temperature relationships. Using sealed vessel reactions normalized to α25 = 1.01025 (the fractionation factor for calcite at 25°C), the apparent fractionation factor at 90°C (α*90) for pristine enamel ranged between 1.00771 and 1.00820, and between 1.00695 and 1.00772 for fossilized enamel. Apparent fractionation factors for common acid bath reactions are similar to those for sealed vessel reactions. A significant correlation exists between α*90 and F content, suggesting that change in the acid fractionation factor may be related to the replacement of OH with F during fossilization of bioapatite. These results have important implications for making accurate comparisons between modern and fossil tooth enamel δ18O values, and for the uniformity of isotope data produced in different laboratories using different acid reaction temperatures. Copyright © 2007 John Wiley & Sons, Ltd.