Estrogens and androgens exert many biological effects that do not require interactions of their receptors with chromosomal DNA. However, it has been a long-standing question how the sex steroid receptors provoke signal transduction outside the nucleus. Here we have shown that epidermal growth factor (EGF) directs sex-specific steroid signaling through Src activation. We have revealed that estrogen (E2)-induced Src activation takes place in, not only plasma, but also endomembranes. This was found ascribed to the existence of EGF and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor together with Src. EGFR, estrogen receptor, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast, the androgen 5␣-dihydrotestosterone-induced Src activation occurs only in the plasma membrane free from the interaction of EGFR with androgen receptor, irrespective of EGF. The cell growth occurred only moderately as a result. The spatial difference in Src activation between E2 and 5␣-dihydrotestosterone may be responsible for the different extent of observed cell growth.Besides traditional genomic pathways of sex steroid receptors in the nucleus, the extranuclear non-genomic pathways of these receptors are being revealed to strongly relate to many biological consequences, including vascular protection and cell proliferation (1-3). These non-genomic pathways are rapidly mediated through several critical protein kinases. A non-receptor protein tyrosine kinase, Src, is known to be activated immediately after a steroid stimulation (4, 5). This activated Src phosphorylates a wide variety of substrate proteins (such as Shc), which finally induce gene transcription (6, 7). In previous studies, the rapid activation of Src and other kinases by steroids has never been demonstrated in living cells.To answer the question about how the Src activity is nongenomically regulated by steroid receptors in single living cells, we developed a fluorescent indicator for Src kinase activity, and named it Srcus. This indicator can monitor substrate phosphorylation by activated endogenous Src as a fluorescent resonance energy transfer (FRET) 5 response in single living cells. Based on the fluorescence imaging with the present fluorescent indicators, we demonstrated that E2-induced Src activation takes place in not only plasma but also endomembranes. This was found ascribed to the existence of epidermal growth factor (EGF) and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor (ER) together with Src. EGFR, ER, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast to estrogen-activated Src signaling, the male steroid hormone 5␣-dihydrotestosterone (DHT) was found to activate Src only in th...