We report the results of a systematic investigation designed to optimize a method for quantifying radioactivity in proteins in sodium dodecyl sulfate-polyacrylamide gels. The method involves dissolving appropriately sized pieces of gel in hydrogen peroxide and heating to 70degreesC overnight followed by liquid scintillation counting. H2O2 had no effect on the count rates of [14 C]bovine serum albumin (BSA) when counted in a conventional liquid scintillation system, and the count rates remained stable for several days. Temperatures below 70degreesC resulted in incomplete extraction of radioactivity from gels containing [C-14]BSA, but there was also a significant reduction in count rates in samples incubated at 80degreesC. At 70degreesC recovery was not affected by the amount of sample loaded onto the gel or by the staining procedure (Coomassie Brilliant Blue or SYPRO Ruby). Recoveries were in the range of 89-94%, and the coefficient of variation for five replicate samples was 5-10%. This method offers a reliable way of measuring the amount of radioactivity in proteins that have been separated by electrophoresis. It may be useful, for example, in quantitative metabolic labeling experiments when it is necessary to know precisely how much tracer has been incorporated into a particular protein.