The oxygen isotope contents (δ18O) of soil, xylem, and leaf water and ecosystem respiration were studied in a ponderosa pine forest during summer 2001. Our goal was to assess whether δ18O of CO2 could be used to quantify the relative contributions of soil and foliar respiration to total nocturnal ecosystem respiration. The δ18O in leaf and soil water showed enrichment over a 2‐week sampling period as the weather became hot and dry (leaves 0.9 to 15.0‰, and soil −10.4 to −3.1‰), while δ18O of xylem water remained constant (−12.9‰). Water in the soil was enriched in 18O near the soil surface (−6.4‰ at 5 cm depth) relative to greater depths (−11.1‰ at 20 cm). The δ18O of ecosystem respiration became gradually enriched over the 2‐week sampling period (from 24.2 initially to 32.9‰ at the end, VSMOW scale). Soil respiration contributed 80 ± 12 percent to the total respiratory flux, close to estimates from scaled‐up chamber data (77% [Law et al., 2001a]). Quantitative application of the isotopic approach to determine respiratory proportions required direct measurement of δ18O of soil and xylem water, air and soil temperature, and humidity. Better estimates of the isotopic signatures of component fluxes could be achieved with additional measurements and more detailed modeling. Results demonstrate that (1) there is variability in δ18O of precipitation inputs to ecosystems, (2) immediately following a precipitation event, δ18O of ecosystem respiration may reflect δ18O of precipitation, (3) periods of hot dry weather can substantially enrich ecosystem water pools and subsequently alter the isotope content of CO2 in ecosystem respiration, and (4) stable oxygen isotopes in CO2 can be used to quantify the foliar and soil components of ecosystem respiration.