Two-dimensional (2-D) gel electrophoresis has been used in conjunction with autoradiography and computerized optical densitometry for quantitating specific protein synthesis. However, accurate quantitation of 2-D autoradiograms requires the prior assessment of such parameters as linearity, reciprocity, and reproducibility. The present study was performed to determine the contribution of each of these to the dissimilation of beta-emission and autoradiographic density, and of density and protein synthesis. Various aliquot volumes of a single complex protein specimen labeled with 35S-amino acids were subjected to 2-D gel electrophoresis, and these gels were serially exposed at graded intervals. The peak densities and volumes of the 111 visualized spots were used to examine the above parameters. In our computerized scanning system, the peak density is a more accurate and reproducible parameter of optical density than is spot volume. Approximately 30% of the dynamic range of peak density is non-linear; quantitation of spots above or below the linear range leads to inaccuracies in quantitation. In addition, the phenomenon of reciprocity, which states that density is directly proportional to exposure (beta-emission of 35S x time), is shown to fail as aliquot volume, or mass of 35S increases. The implications of reciprocity failure to accurate quantitation are discussed. Finally, the sources of variance in autoradiographic analysis were examined, by assessing the intra-scan, intra-gel run, and inter-gel run coefficients of variation. The results of this study show that autoradiographic densitometry is an effective method for quantitation of 2-D gels, but linearity, reciprocity, and reproducibility must be assessed prior to its experimental use. Restrictions of such use are suggested.