Animals were exposed to nitric oxide (NO) in order to study its biological effect. First a method was developed to exclude nitrogen dioxide formed during exposure of NO. Nitrosyl-hemoglobin (NOHb) in the blood of mice, rats, and rabbits exposed to NO was determined by electron spin resonance spectrometry at -100C. The content of NOHb in the blood of the mouse reached the maximum (0.13% of the total hemoglobin) 20 minutes after the exposure was begun and thereafter a state of equilibrium was maintained. The NOHv level decreased to one half in several minutes after the mouse was removed to a filtered air environment. Dose-response relationship was observed between gas concentrations and the maximum NOHb level.