The oxygen and hydrogen isotopic composition of Eocene and Miocene freshwater cherts in the western United States records regional climatic variation in the Cenozoic. Here, we present isotopic measurements of 47 freshwater cherts of Eocene and Miocene age from the Great Basin of the western United States at two different sites and interpret them in light of regional climatic and tectonic history. The large range of ␦ 18 O of terrestrial cherts measured in this study, from 11.2‰ to 31.2‰ (SMOW: Standard Mean Ocean), is shown to be primarily the result of variations in ␦ 18 O of surface water. The following trends and patterns are recognized within this range of ␦ 18 O values. First, in Cenozoic rocks of northern Nevada, chert ␦ 18 O records the same shift observed in authigenic calcite between the Eocene and Miocene that has been attributed to regional surface uplift. The consistent covariation of proxies suggests that chert reliably records and retains a signal of ancient meteoric water isotopic composition, even though our analyses show that chert formed from warmer waters (40°C) than coexisting calcite (20°C). Second, there is a strong positive correlation between ␦ 18 O and ␦D in Eocene age chert from Elko, Nevada and Salina, Utah that suggests large changes in lake water isotopic composition due to evaporation. Evaporative effects on lake water isotopic composition, rather than surface temperature, exert the primary control on the isotopic composition of chert, accounting for 10‰ of the 16‰ range in ␦ 18 O measured in Eocene cherts. From authigenic mineral data, we calculate a range in isotopic composition of Eocene precipitation in the north-central Great Basin of Ϫ10 to Ϫ14‰ for ␦ 18 O and Ϫ70 to Ϫ100‰ for ␦D, which is in agreement with previous estimates for Eocene basins of the western United States. Due to its resistance to alteration and record of variations in both ␦ 18 O and ␦D of water, chert has the potential to corroborate and constrain the cause of variations in isotope stratigraphies.