The analysis of the absorption spectra of model compounds of tyrosine and phenylalanine residues by means of fourth-derivative spectrophotometry is able to separate the contribution of the two chromophores, thus allowing the study of each one. Fourth-derivative analysis resolves the two main vibrational bands of tyrosine, giving rise to two peaks which are sensitive to changes in the environment of the phenolic ring. The parameters obtained from the fourth-derivative spectra were found to depend on the strength of the hydrogen bonds formed by the OH group of tyrosine, as well as on the heterogeneity of tyrosine environments. It is also shown that the fourth-derivative tyrosine peaks are not perturbed by broad bands, such as that arising from ionized tyrosine chromophores. The peaks arising from the phenylalanine model, although less sensitive than those of tyrosine, were found to depend on the polarity of the environment. As a check of the method, it is applied to the study of tyrosine and phenylalanine residues of calf thymus histone H1 and bovine pancreatic ribonuclease A.