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Isoflurane has been characterized as a distressing agent for rodents, causing both physiologic and behavioral effects. Using a "darkened home cage" has been recommended during CO2 administration for rodent euthanasia; this is arguably a similar animal experience to anesthetic induction with isoflurane. Based on the premise that rodents perceive red light as darkness via the primary optic tract, we compared physiologic and behavioral markers of stress in 2 inbred strains of mice (C57BL/6J and BALB/cJ) anesthetized with isoflurane in either a red-tinted (dark) induction chamber or a traditional translucent induction chamber. Physiologic stress was assessed based on plasma levels of norepinephrine, epinephrine, and corticosterone. Stress-related behaviors (rearing, face wiping, and jumping) were recorded on video and scored from initiation of induction to loss of consciousness. No significant correlations were found between chamber type and physiologic stress hormones. As compared with the translucent chamber, stress-related behaviors were more frequent in the red-tinted chamber, including: 1) significantly higher rearing frequencies in BALB/cJ mice; 2) higher behavioral stress scores in BALB/cJ and male C57BL/6J mice; and 3) more face wiping behavior when considering all mice combined. These findings suggest that mice do not experience significant alleviation of physiologic indices of stress when anesthetized in a red-tinted induction chamber. Furthermore, isoflurane induction in the red-tinted chamber appeared to increase the expression of stress-related behaviors, particularly in BALB/cJ mice. Based on our findings and a growing body of literature on the unintended effects of red light, we do not recommend using red-tinted chambers for induction of anesthesia in mice.