Abstract Information about climatic conditions during plant growth is preserved by the oxygen-isotope composition of biogenic silica (phytoliths) deposited in grasses. The oxygen-isotope composition of phytolith silica is dependent on soil-water δ 18O values, relative humidity and evapotranspiration, and temperature during plant growth. Phytolith and plant-water δ 18O values for C3 ( A. breviligulata) and C4 ( C. longifolia) grasses from natural and greenhouse sites in southwestern Ontario were used to compare the isotopic fractionation between biogenic silica and water in various parts of these living plants. For non or weakly transpiring tissues (rhizomes, stems, sheaths) in both grass species, the Δ 18O silica-plant water remained constant at ∼34‰, and the δ 18O and δD values of plant water collected from pre-dawn and mid-day samplings showed little variation. These plant waters were only slightly enriched in 18O and D relative to water provided to the grasses. Isotopic temperatures calculated from the silica and plant-water isotopic data matched measured growing temperatures for the region. By comparison, the upper leaf water was extremely enriched in oxygen-18 and deuterium at maximum rates of transpiration relative to water from non-transpiring tissues, as were the calculated, steady-state values for leaf-water δ 18O and δD. Silica produced in the transpiring tissues (leaf, inflorescence) has higher δ 18O values than silica from non-transpiring tissues, but the enrichment is modest compared to upper leaf water under mid-day conditions. Leaf phytoliths have formed from plant water typical of average conditions in the lower leaf, where the extreme 18O-enrichment is not encountered. C. longifolia was also collected from Alberta and Nebraska, where growing conditions are different from southwestern Ontario. Phytoliths at all three sites have a similar pattern of δ 18O values within the plants, but the isotopic separation between leaf and stem silica increases from 4 to 8‰ as average relative humidity decreases. The difference between actual growing temperature and that calculated using measured δ 18O values for stem silica and local meteoric water became progressively larger as relative humidity decreased, likely because of evaporative 18O-enrichment of soil water. Such effects are most pronounced in arid environments and pertinent in grasslands where much of the active rooting zone can be situated at the shallower depths most affected by the 18O-enrichment of soil water.