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In semi-arid regions, where plants using both C-3 and C-4 photosynthetic pathways are common, the stable C isotope ratio (delta C-13) of ecosystem respiration (delta C-13(R)) is strongly variable seasonally and inter-annually. Improved understanding of physiological and environmental controls over these variations will improve C cycle models that rely on the isotopic composition of atmospheric CO2. We hypothesized that timing of precipitation events and antecedent moisture interact with activity of C-3 and C-4 grasses to determine net ecosystem CO2 exchange (NEE) and delta C-13(R). Field measurements included CO2 and delta C-13 fluxes from the whole ecosystem and from patches of different plant communities, biomass and delta C-13 of plants and soils over the 2000 and 2001 growing seasons. NEE shifted from C source to sink in response to rainfall events, but this shift occurred after a time lag of up to 2 weeks if a dry period preceded the rainfall. The seasonal average of delta C-13(R) was higher in 2000 (-16aEuro degrees) than 2001 (20aEuro degrees), probably due to drier conditions during the 2000 growing season (79.7 mm of precipitation from April up to and including July) than in 2001 (189 mm). During moist conditions, delta C-13 averaged -22aEuro degrees from C-3 patches, -16aEuro degrees from C-4 patches, and -19aEuro degrees from mixed C-3 and C-4 patches. However, during dry conditions the apparent spatial differences were not obvious, suggesting reduced autotrophic activity in C-4 grasses with shallow rooting depth, soon after the onset of dry conditions. Air and soil temperatures were negatively correlated with delta C-13(R); vapor pressure deficit was a poor predictor of delta C-13(R), in contrast to more mesic ecosystems. Responses of respiration components to precipitation pulses were explained by differences in soil moisture thresholds between C-3 and C-4 species. Stable isotopic composition of respiration in semi-arid ecosystems is more temporally and spatially variable than in mesic ecosystems owing to dynamic aspects of pulse precipitation episodes and biological drivers.