CCooled oxygen inhalation was hypothesized as a novel hypothermia technique in a previous study. In the current study, we aimed to determine the optimal temperature of oxygen for this method. This is a prospective, randomized, controlled, examiner-blinded experimental study conducted with 45 healthy, adult, Wistar Hannover male rats. Rats were randomly divided into five groups; group 1: +4 degrees C intubated group (n = 7), group 2: +4 degrees C nonintubated group (n = 9), group 3: +8 degrees C intubated group (n = 9), group 4: +8 degrees C nonintubated group (n = 9), and group 5: control group (n = 9). The control group received only a standardized anesthesia protocol, and no hypothermia technique was administered. Cooled oxygen was administered in the four study groups until the rectal temperature reached 34 degrees C. The target temperature was maintained between 32 degrees C and 34 degrees C for 2 hours. Then, hypothermia protocols were terminated and rats were rewarmed externally with a blanket. Main outcomes were the speed (degrees C/minute) of temperature decrease (S) and the time required to reach the target body temperature (T). All study groups had better results than the control group in T and S values (p < 0.001) for both parameters. Group 1 had a better T value than group 4 (p = 0.01), but no difference in S value (p = 0.223). Comparison of group 2 and group 4 showed that group 4 had better results in T and S (p = 0.04 and 0.001, respectively). No pathologic changes in histologic examination were observed in any group. Our study showed that the optimal temperature of oxygen for the cooled oxygen technique was +4 degrees C through an intubation tube.