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We investigate how oxygen isotopes in equid teeth can be used as a record of seasonality. First, we use in situ laser ablation and conventional microsampling techniques to understand time-averaging of environmental signals in intra-tooth isotope profiles in modern feral horse teeth (n = 5) from Mongolia, where there is a large seasonal gradient in the oxygen isotopic composition of precipitation. We demonstrate that laser ablation can be used to sample inner, middle, and outer enamel layers in large mammalian herbivore teeth. The inner enamel layer records less attenuated isotopic signals than other layers, as predicted by the mineralization patterns, but intra-tooth signal amplitude is similar for laser and conventional sampling methods. Second, we use modern zebra teeth (n = 21) collected in eastern Africa to evaluate how intra-tooth oxygen isotope variation relates to rainfall patterns in the tropics. We show that the intra-tooth isotopic range increases with intra-annual precipitation isotopic range, which in turn relates to aridity in the equatorial bimodal rainfall region but is influenced by other hydroclimate processes in the region as a whole. Finally, we address isotopic seasonality during the Early Pleistocene in eastern Africa using oxygen isotopes in fossil equid teeth from southwestern Kenya (n = 11) and northern Tanzania (n = 5). We find variable isotopic seasonality in the past, similar to present-day eastern Africa, consistent with the notion that hominins and other mammals were able to accommodate environmental variability on intra-annual scales in addition to well-documented orbital cycles. [ABSTRACT FROM AUTHOR]