Soil respiration is the major pathway of carbon transfer from soil to atmosphere. Soil CO2 has an important influence on carbon and oxygen isotopic composition of atmospheric CO2. The objectives of this study were to trace seasonal changes in oxygen isotopic composition of soil CO2, to test if this diffusional effect was constant and to evaluate if factors other than diffusion also affected [delta]18O values of soil CO2, including soil water, CO2 production and different species (C3 and C4). To achieve these goals, soil CO2 samples were collected from chambers free of roots (RF) and from chambers containing roots (WR). Samples collected first were called Sample I, and Sample II referred to those collected second. Oxygen isotope results for the collected samples indicated that soil CO2 isotopic values for WR and RF chambers were statistically undistinguishable, suggesting that soil CO2 and soil water were likely to be in isotopic equilibrium. Similar to other findings, these results also indicated that soil water played the most important role on setting oxygen isotopic signature of soil CO2. Because CO2 production was different in WR and RF chambers, the similarity in isotopic values found in these two different chambers also indicated that soil CO2 production had no observable impact on oxygen isotopic composition of soil CO2. Similarly, there was no significant difference in isotopic values for soil CO2 in soil containing C3 and C4 plants, suggesting that different species had a negligible effect on respired CO2; however, a statistically significant difference of [Difference]2% was found between Sample I and Sample II collected over the 2003 growing season and April 2004. This difference was interpreted to result from different diffusional effects of soil CO2 that caused different kinetic enrichment factors. Rainwater [delta]18O values and soil temperature were employed to estimate the kinetic effect of diffusion on oxygen isotopic composition of soil CO2. It was found that the kinetic enrichment factor varied through time, implying that the use of a constant value for this factor likely affected global estimations of oxygen isotopic composition of soil CO2.