The molar absorption coefficient, ε, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring ε for a protein.(This method is described by Gill and von Hippel [1989, Anal Biochem 182: 319–326] and is based on data from Edelhoch [1967, Biochemistry 6: 1948–1954].) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average ε values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the ε values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average ε values for the proteins are less than the ε values measured in any of the solvents. For Tyr, the average ε values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured ε values for 80 proteins, the ε at 280 nm of a folded protein in water, ε (280), can best be predicted with this equation