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We conducted high frequency measurements of the δ¹⁸O value of atmospheric CO₂ from a juniper (<i>Juniperus monosperma</i>) woodland in New Mexico, USA, over a four-year period to investigate climatic and physiological regulation of the δ¹⁸O value of ecosystem respiration (&delta;_R). Rain pulses reset &delta;_R with the dominant water source isotope composition, followed by progressive enrichment of &delta;_R. Transpiration (<i>E</i>_T) was significantly related to post-pulse &delta;_R enrichment because the leaf water δ¹⁸O value showed strong enrichment with increasing vapor pressure deficit that occurs following rain. Post-pulse &delta;_R enrichment was correlated with both <i>E</i>_T and the ratio of <i>E</i>_T to soil evaporation (<i>E</i>_T/<i>E</i>_S). In contrast, the soil water δ¹⁸O value was relatively stable and &delta;_R enrichment was not correlated with <i>E</i>_S. Model simulations captured the large post-pulse &delta;_R enrichments only when the offset between xylem and leaf water δ¹⁸O value was modeled explicitly and when a gross flux model for CO₂ retro-diffusion was included. Drought impacts &delta;_R through the balance between evaporative demand, which enriches &delta;_R, and low soil moisture availability, which attenuates &delta;_R enrichment through reduced <i>E</i>_T. The net result, observed throughout all four years of our study, was a negative correlation of post-precipitation &delta;_R enrichment with increasing drought.