At research institutions, isoflurane delivered by precision vaporizer to a face mask is the standard for rodent surgery and for procedures with durations that exceed a few minutes. Pure oxygen is often used as the carrier gas for isoflurane anesthesia, despite documented complications from long-term 100% oxygen use in humans and known occupational safety risks. We therefore examined the effect of anesthetic delivery gas on physiologic variables in mice and rats. Rodents were anesthetized for 60 min with isoflurane delivered in either 21% or 100% oxygen by means of a nose cone. We noted no difference between carrier gasses in physiologic variables in mice, including body temperature, respiratory rate, mean arterial pressure, surgical recovery time, pH, or PaCO2. However, blood gas analysis revealed evidence of a ventilation-perfusion mismatch in the 100% oxygen group. Pressure-volume hysteresis and histomorphometric analyses confirmed the presence of increased atelectasis in mice that received 100% oxygen. Unlike mice, rats that received isoflurane in 100% oxygen had acute respiratory acidosis and elevated mean arterial pressure, but atelectasis was similar between carrier gasses. Our data suggest that both 100% and 21% oxygen are acceptable for the delivery of isoflurane to mice. However, mice anesthetized for studies focused on lung physiology or architecture would benefit from the delivery of isoflurane in 21% oxygen to reduce absorption atelectasis and the potential associated downstream inflammatory effects. For rats, delivery of isoflurane in 21% and 100% oxygen both caused perturbations in physiologic variables, and choosing a carrier gas is not straightforward.