Isoflurane (ISO) is the most commonly used inhalational anesthetic for experimental interventions in mice and is preferred for imaging technologies that require the mouse to remain anesthetized for relatively long time periods. This study compares the stability of mean arterial pressure (MAP), heart rate (HR), and body temperature under ISO concentrations of 1%, 1.5%, and 2%(volume-to-volume, v/v) for up to 90 minutes postinduction. At all three levels of anesthesia, we examined evoked physiological responses to fractional inspiratory ratio variations of oxygen (FiO 2) and nitrous oxide (N 2 O). In addition, we determined the hemodynamic effects of anesthesia on pH, glucose, insulin, glucocorticoids, and partial pressure of oxygen and of carbon dioxide in the blood (p a O 2, p a CO 2). The results indicate that the most appropriate ISO dose level was 1.5% v/v, yielding stable MAP and HR values comparable to those observed in the animal’s conscious state, with a minute-to-minute variability in MAP and HR of≤ 11%. Based on such recordings, the optimal FiO 2 appeared to be 50%. The additional use of N 2 O was associated with higher and more stable values of MAP and HR. Arterial pH values were within the physiological range and varied between 7.20 and 7.43. ISO anesthesia at 1.5% v/v was also associated with mild hyperglycemia (+ 47%), whereas insulin levels and corticosteroids remained unaltered. We conclude that the application of isoflurane as an inhalational anesthetic in the mouse can be optimized to attain stable hemodynamics by administering it at 1.5% v/v and by supplementing it with N 2 O.