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The calibration method described is based on the in situ formation of carbon monoxide in a closed system by the reaction of hot concentrated sulfuric acid with formic acid. Carbon monoxide is released from hemoglobin by treatment with 85% phosphoric acid, diluted 1:2 (v/v). Carbon monoxide is analyzed by means of headspace gas chromatography on a Porapak Q 80–100 column, following catalytic reduction to methane, using a flame-ionization detector. The method was validated by comparing the results obtained for blood samples from patients suffering from carbon monoxide poisoning and previously analyzed by means of spectrophotometry.