AbstractThe two‐wavelength method is the microspectrophotometric method of choice for thick specimens. Its accuracy is dependent on the accuracy of selection of the operating wavelength‐pair. The “slope zero test,” offered as a check upon the accuracy of this selection (Garcia and Iorio, ′66) is shown to be invalid by analysis of experimental data, by computer model and by mathematical derivation. A means of checking the accuracy of selection of the operating wavelength‐pair via scanning measurements is suggested. A new formulation of the two‐wavelength method, permitting use of different operating wavelength‐pairs, is also given.