A method which utilizes computerized microdensitometry to measure amino acid compositions of polypeptides resolved in two-dimensional gels is described. Twenty cultures of two human cell lines were radiolabeled with each of the 20 different 14C or 35S – amino acids. Two-dimensional polyacrylamide gel electrophoresis was performed on total protein samples of the cultures in replicates of three. For each amino acid label the relative amount of radioactivity in an “unknown” polypetide species was computed using the measured radioactivity simultaneously incorporated into the reference protein, β-, γ-actin, whose precise amino acid composition is known. We tested the accuracy of this method by computing the amino acid composition of two species of mutant β-actin, which along with normal β-, γ-actin as the reference provided us with an ideal set of conditions. We found that amino acid composition can be predicted with a standard error of 6% over all twenty amino acids. Techniques are described to further reduce the measurement error so that compositions of proteins can be measured under less ideal conditions.