We have measured, by double-label SDS-elecirophoresis, the effect of estradiol on the relative rates of specific protein synthesis in MCF-7 human breast cancer cells. Two specific proteins, a 24,000 molecular weight (24k) cytosol protein and a 54,000 molecular weight (54k) protein located in nuclei, were found to be preferentially stimulated by estradiol. Estrogen stimulation of the 24k protein has been characterized under various experimental conditions. Synthesis was found to increase with time of estrogen incubation, reaching maximum stimulation between 4 and 6 days, was estrogen dose-dependent within the physiological range of hormone concentration and stimulation required specifically those steroids which bind to the estrogen receptor. Moreover, the magnitude of increased 24k synthesis parallels nuclear estrogen receptor processing, an event correlated with another estrogen response in MCF-7, induction of progesterone receptor. Double-label cytosols were further resolved by two-dimensional electrophoresis, which revealed two estrogen stimulated proteins in the 24k region with isoelectric points in the range of 6.7–6.9 and 6.4–6.7. The more acidic species was determined to represent a major fraction (1.6%) of newly synthesized protein. Increased 54k synthesis is detected only in nuclei and this response to estrogen has not yet been fully characterized. A 24k and a 54k protein have been synthesized in a rabbit reticulocyte translation system with MCF-7 mRNA. Adapting the double-label electrophoresis technique to this cell-free system, estradiol was found also to preferentially stimulate translatable 24k and 54k mRNA activity, suggesting that estradiol effects 24k and 54k synthesis at the transcriptional level.