A new method of determination of COHb is reported in this paper by two- wavelength second-derivative spectrophotometry.Blood was diluted with a solution of THAM containing Na_2S_2O_4.The second-derivative spectrum was measured in the region of 500-600 nm and the derivative absorbance differerce ?Ax was deter mined at the wavelengths of 564 nm and 576 nm.Then the solution was satura- ted with CO gas.The second-derivative spectrum of COHb was measured in the same region.The derivative absorbance difference ?A_(100) was determined at the same wavelengths.The COHb in blood was calculated by the ratio of ?Ax to ?A_(100).The results of determination of COHb in standard blood samples were in good agreement with the theoretical values.