Previous studies have shown that synthesis of a 24,000 molecular weight (24k) protein (7) and the mRNA activity coding for a 24k protein (8) are selectively stimulated by estradiol in MCF-7 human breast cancer cells. Utilizing a double-label electrophoresis technique to measure the relative rates of specific protein synthesis, the present study relates further characterization of this estrogen response. The 24k protein was found to be stimulated by estradiol provided cells were preincubated with antiestrogen. Under these conditions, antiestrogens inhibit cell growth and addition of estradiol reverses growth inhibition (estrogen growth ‘rescue’). As a control experiment, specific protein synthesis was measured in another growth rescue model produced by withdrawing serum from the medium to inhibit growth and then stimulating cells by readdition of serum (serum growth ‘rescue’). This failed to stimulate synthesis of a 24k protein, implying that the effect observed following estrogen ‘rescue’ was not a non-specific result of growth stimulation. Increased 24k synthesis was found to be both time and estrogen-dose dependent and required specifically those steroid hormones which interact with the estrogen receptor. Moreover, the dose response curve for 24k stimulation paralleled closely the dose response for estrogen stimulation of nuclear estrogen receptor processing, an event correlated with another estrogenic response in MCF-7 cells, induction of progesterone receptor. These findings suggest that estradiol itself directly stimulates synthesis of the 24k protein through interaction with the estrogen receptor system. Further separation of double-label cytosols by two-dimensional electrophoresis resolved the 24k protein into two isoelectric species with pI's in the range of 6.7–6.9 and 6.4–6.7, the latter being the most abundant or rapidly labeling protein. Based on the percentage of total cytosol incorporation contained in individual two-dimensional gel spots, the major species of 24k represented in the fully stimulated cell (3H-counts) about 1.6% of newly synthesized protein. It also represented about 0.7% of newly synthesized protein in the unstimulated (14C-counts) cell, which indicates that 24k is synthesized constitutively. Because of its relative abundance, the 24k protein may be a suitable end product for investigating the mechanisms of estrogen action in human breast cancer.