The presence of large amounts of a 67 kDa estradiol receptor that does not bind hormone was observed in 8 to 37 human mammary tumors (34 malignant and 3 benign). This form of receptor was detected by its conversion to hormone binding receptor by an endogenous tyrosine kinase in vitro. All 8 tumors were malignant. In these, the incubation of cytosol with ATP was seen to cause a 1- to 5-fold increase in estradiol-specific binding sites. These sites bound estradiol with physiological affinity, and their appearance was associated with tyrosine phosphorylation of estradiol receptor. The enzyme converting the non-hormone binding receptor into the hormone binding receptor is largely present in cytosol and scarce in membranes. It has been extensively purified. It is a 67 kDa protein under denaturating conditions, binds calmodulin-Sepharose in a Ca2+-dependent manner, is stimulated by Ca2+ and calmodulin, phosphorylates exogenous actin, is activated by the estradiol-receptor complex. The enzyme interacts with antibodies directed against the carboxy-terminal and catalytic domains of c-src. Therefore, it is a putative new member of the large c-src-related kinase family. Human mammary cancers with significant amounts of 67 kDa non-hormone binding receptor show relatively low levels of hormone binding estradiol receptor. The presence of non-hormone binding receptor that can be activated by in vitro tyrosine phosphorylation suggests that functional interaction of estradiol receptor with tyrosine kinases is altered in malignant tumors and has bearing on loss of hormone dependence and progression of the mammary cancer malignancy.