How the isotopic composition of the teeth of slow-growing primates (including humans) reflects the environmental water supply is still poorly understood, despite its potential for inferring seasons of birth and death, historic and prehistoric rainfall patterns, and sustained droughts and extreme rainfall events. Here we compare secondary ion mass spectrometry (SIMS) measurements of δ18O values in the innermost enamel of a human child and three wild chimpanzees (Pan troglodytes verus) with concurrent local rainfall. We also compare enamel δ18O values from ten captive macaques (Macaca mulatta) to their drinking water δ18O values and/or concurrent rainfall. Synchrotron X-ray imaging shows that the innermost ∼20 µm of enamel is highly mineralized soon after secretion. SIMS microsampling of this zone yields a coherent record of local rainfall patterns—lower δ18O values during wet seasons, higher values during more arid periods, which is particularly evident in wild chimpanzees. Cross-correlation analyses reveal statistically significant relationships between enamel δ18O values and water inputs that are strongest in healthy individuals without physiological disruptions. Primate enamel δ18O values commonly vary by ∼1–2 ‰ within a few weeks of birth, as well as in captive macaques during periods of dehydration due to diarrheal illnesses. Consumption of cooked foods and manipulation of municipal water supplies may also obscure rainfall trends in contemporary humans and captive primates, although several healthy macaques show parallels with δ18O values measured from drinking water. Predicted drinking water δ18O values (δ18Odw) from existing water/phosphate conversion equations reveal a strong similarity with observed δ18Odw values, including measured meteoric and tap water. While enamel maturation may impact primate δ18O values, our analysis does not detect consistent temporal offsets between physiological events or seasons and the innermost enamel. These data show that SIMS δ18O measurements of living and fossil primate enamel can provide a high-fidelity record for paleoseasonality reconstruction.