We tested the hypothesis that the stable carbon isotope signature of ecosystem respiration (δ13CR) was regulated by canopy conductance (Gc) using weekly Keeling plots (n=51) from a semiarid old-growth ponderosa pine (Pinus ponderosa) forest in Oregon, USA. For a comparison of forests in two contrasting climates we also evaluated trends in δ13CR from a wet 20-year-old Douglas-fir (Pseudotsuga menziesii) plantation located near the Pacific Ocean. Intraannual variability in δ13CR was greater than 8.0‰ at both sites, was highest during autumn, winter, and spring when rainfall was abundant, and lowest during summer drought. The δ13CR of the dry pine forest was consistently more positive than the wetter Douglas-fir forest (mean annual δ13CR: −25.41‰ vs. −26.23‰, respectively, P=0.07). At the Douglas-fir forest, δ13CR–climate relationships were consistent with predictions based on stomatal regulation of carbon isotope discrimination (Δ). Soil water content (SWC) and vapor pressure deficit (vpd) were the most important factors governing δ13CR in this forest throughout the year. In contrast, δ13CR at the pine forest was relatively insensitive to SWC or vpd, and exhibited a smaller drought-related enrichment (∼2‰) than the enrichment observed during drought at the Douglas-fir forest (∼5‰). Groundwater access at the pine forest may buffer canopy–gas exchange from drought. Despite this potential buffering, δ13CR at the pine forest was significantly but weakly related to canopy conductance (Gc), suggesting that δ13CR remains coupled to canopy–gas exchange despite groundwater access. During drought, δ13CR was strongly correlated with soil temperature at both forests. The hypothesis that canopy-level physiology is a critical regulator of δ13CR was supported; however, belowground respiration may become more important during rain-free periods.