Abstract This lab previously showed that brief inhalation of high concentrations of CO 2 results in a prolonged, moderate antinociception with characteristics of a nonopiate, hormonal mechanism. To further characterize and optimize this response, the effect of a variety of methodological, biological, and stress-related manipulations were studied. No significant differences were found in the CO 2-induced response between animals that were tested during different portions of their diurnal cycles, in rats that were unhandled or habituated to nociceptive testing conditions, in male vs. female rats, or in animals of differing weights. Additionally, restraining animals prior to CO 2 exposure induced a hot plate antinociceptive response that was not different from the response produced by CO 2 alone. In contrast, on the tail flick test, a CO 2 -restraint interaction both increased and decreased the response at different times. The present findings show that CO 2 antinociception: a) is a reliable phenomenon not altered by a variety of methodological and biological conditions, and b) has characteristics of a novel, stress-mediated antinociceptive response.