Chamber measurements of total ecosystem respiration (TER) in a native Canadian grassland ecosystem were made during two study years with different precipitation. The growing season (April–September) precipitation during 2001 was less than one-half of the 30-year mean (1971–2000), while 2002 received almost double the normal growing season precipitation. As a consequence soil moisture remained higher in 2002 than 2001 during most of the growing season and peak aboveground biomass production (253.9 g m−2) in 2002 was 60% higher than in 2001. Maximum respiration rates were approximately 9 μmol m−2 s−1 in 2002 while only approximately 5 μmol m−2 s−1 in 2001. Large diurnal variation in TER, which occurred during times of peak biomass and adequate soil moisture, was primarily controlled by changes in temperature. The temperature sensitivity coefficient (Q10) for ecosystem respiration was on average 1.83 ± 0.08, and it declined in association with reductions in soil moisture. Approximately 94% of the seasonal and interannual variation in R10 (standardized rate of respiration at 10 °C) data was explained by the interaction of changes in soil moisture and aboveground biomass, which suggested that plant aboveground biomass was good proxy for accounting for variations in both autotrophic and heterotrophic capacity for respiration. Soil moisture was the dominant environmental factor that controlled seasonal and interannual variation in TER in this grassland, when variation in temperature was held constant. We compared respiration rates measured with chambers and that determined from nighttime eddy covariance (EC) measurements. Respiration rates measured by both techniques showed very similar seasonal patterns of variation in both years. When TER was integrated over the entire growing season period, the chamber method produced slightly higher values than the EC method by approximately 4.5% and 13.6% during 2001 and 2002, respectively, much less than the estimated uncertainty for both measurement techniques. The two methods for calculating respiration had only minor effects on the seasonal-integrated estimates of net ecosystem CO2 exchange and ecosystem gross photosynthesis.