The precise role of src-type kinases as signal transducers has been under intensive investigation but only in a few instances has their role been revealed in any detail. Thus, src, fyn and yes are activated upon stimulation by platelet-derived growth factor or colony-stimulating factor in cells expressing high levels of these receptors. Activation of src-family kinases by other receptor tyrosine kinases such as the epidermal-growth-factor (EGF) receptor has not been directly demonstrated. In this report, we demonstrate EGF-dependent activation of src-family tyrosine kinases in NIH3T3 cells overexpresssing the human EGF receptor. Activation is rapid (<1 min) and persistent (up to 16 h). Furthermore, we show a correlation between the level of EGF receptor expressed and the degree of src-family kinase activation. We show that src-family kinase activity is also activated by addition of EGF to PC12 cells, which endogenously express relatively high levels of EGF receptor. Most strikingly, we show that A431 cells, which endogenously express very high levels of EGF receptor, show 10-fold elevated src-family kinase activity as compared to DHER14 cells, and that this activity is constitutive. This activity is completely blocked by AG1478, a specific inhibitor of the EGF-receptor tyrosine kinase activity, pointing to a direct link between overexpression of the EGF receptor and enhanced src-family kinase activity. Our findings suggest that EGF-dependent src-family kinase activity is detectable only when the levels of EGF receptor reach a specific level. Additionally, high levels of EGF receptor, as in A431 cells, may contribute to the elevated activation of src-family kinases. Sustained src-family kinase activation, similar to that seen in v-src-trans-formed cells, may play a role in tumorogenesis and tumor maintenance.