The neurotransmitters and receptor types involved in the afferent arm of the human pupillary light reflex are unknown. We hypothesized that the pupillary light reflex is mediated in part by NMDA receptors and that it would be depressed by the NMDA antagonists, nitrous oxide and ketamine. To study this question, sixteen patients received general anesthesia with desflurane, fentanyl, and muscular relaxation with rocuronium. After a stable level of general anesthesia had been obtained and at least 1h after the start of the surgical procedure, ketamine 1mg/kg (N=8) or saline (N=8) was injected intravenously by random selection. Heart rate, pupil size, pupillary light reflex, BIS scores, and blood pressure were measured every 2min before and for 30min after drug administration. A similar study of sixteen patients was then conducted with either addition of 60% nitrous oxide or 60% nitrogen to the gas mixture. We observed that the pupillary light reflex was depressed by ketamine and nitrous oxide by approximately 50%. The BIS score, representing the processed electroencephalogram, was elevated by ketamine and unchanged with nitrous oxide. Heart rate, pupil size, and blood pressure were unchanged by the drugs when compared to the control groups. We conclude that the two NMDA antagonists ketamine and nitrous oxide depress the human pupillary light reflex during general anesthesia whereas other monitored parameters were either unchanged or paradoxically elevated by the drugs. These findings present evidence that glutamate NMDA receptor activation is involved in generating the human pupillary light reflex.