The primary objective of this study was to compare the effect of 2 commonly used anesthetics, isoflurane and CO2, on the physiologic stress hormone, corticosterone, in rats during serial blood collections. Circulating corticosterone concentrations were monitored during serial jugular blood sampling in rats exposed to either isoflurane or CO2 anesthesia. Blood was drawn under anesthesia at 6 time points (initial sampling and 0.5, 1, 2, 3, and 24 h after initial sampling) across a 24-h period. The results indicated that corticosterone levels in both anesthesia groups showed a similar pattern of stimulation, indicated by a sharp increase in circulating concentrations by the 0.5-h time point, with further elevation at 1 h, and a decline at subsequent time points. Isoflurane-treated animals showed higher baseline levels of corticosterone at the initial sampling. However, the increase in corticosterone at subsequent time points was significantly higher for animals exposed to CO2, suggesting that the cumulative effects of repeated exposure to stressful stimuli was more evident for this type of anesthesia. The apparent reduction in stress effects of isoflurane as opposed to CO2, coupled with other published adverse effects of CO2, suggests that isoflurane is the better choice when anesthesia is needed for serial blood collection. However, availability of equipment for delivery of anesthetic, scavenging of waste gasses, familiarity of personnel with the anesthetic, and the potential effect of the anesthetic on research endpoints should all be considered in choosing between the 2 anesthetics.