Estradiol stimulates protein phosphorylation on tyrosine in human breast cancer MCF-7 cells under conditions of estradiol-stimulated cell growth. The stimulatory effect of estradiol has been observed by 32P-labeling of cells followed by purification of proteins using antiphosphotyrosine antibody coupled to agarose and confirmed by immunoblotting analysis with antiphosphotyrosine antibody. This stimulation is immediate (maximal in 10 s) and transient. In addition, it is receptor-mediated since estradiol stimulation is prevented by two well-known antiestrogens, OH-Tamoxifen and ICI 164,384. Estradiol fails to stimulate tyrosine protein phosphorylation of Cos cells which do not express the estradiol receptor. Two substrates of the estrogen stimulated phosphorylation on tyrosine with approximate mol wt of 55 and 60 kDa interact with a polyclonal antibody raised against amino acids 527-533 of pp60c-src (anti-cst.1 antibody). Tyrosine kinase activity of immunoprecipitates made using either anti cst.1 antibody or the monoclonal 327 antibody specific for pp60c-src shows that kinase(s) strongly related to pp60c-src are immediately and transiently stimulated by estradiol treatment of cells. The present findings provide the first demonstration that a steroid hormone rapidly stimulates tyrosine phosphorylation of target cells and induces functional modifications of substrates of this phosphorylation. These modifications might initiate the estradiol action on cell growth.