OBJECTIVE To investigate factors affecting the determination of the percent carboxyhemoglobin saturation (HbCO%) of blood in an attempt to offer further data for results interpretation and sample storage requirement. METHODS The HbCO% of blood samples stored in various conditions were detected by three spectrophotometries during the succeeding 30 days. RESULTS The data detected by reductive double-wavelength spectrophotometry and double-wavelength spectrophotometry were more stable than mono-wavelength spectrophotometry. The HbCO% of blood was significantly related with the storage conditions which include temperature, time and the degree of exposure to air. CONCLUSIONS Determinations of HbCO% are reliable which performed by reductive double-wavelength spectrophotometry and double-wavelength spectrophotometry, combine with spectral scans. During 30 days, blood stored at 4 degrees C exposed to a limited volume of air does not influence the determination of HbCO%.