The Epidermal Growth Factor Receptor (EGF-R) becomes constitutively tyrosine phosphorylated on two novel sites in v-Src transformed cells, and these phosphorylations are associated with enhanced signaling activity . To determine whether Src could directly phosphorylate these sites, we have examined the ability of the Src kinase to phosphorylate both wild-type and kinase-defective EGF-Rs in vitro. Although purified Src could phosphorylate EGF-Rs, the pattern of phosphorylation sites was not identical to what was previously found in vivo : Src in vitro directly phosphorylated EGF-Rs on one autophosphorylation site (Tyr 1173) which was not a site of re-induced in vivo phosphorylation, suggesting the in vivo inaccessibility of this site. One Src-specific in vitro phosphorylation site (Tyr 03) appeared to correspond to one of the in vivo Src-induced sites (sPY2), but the other Src-specific in vivo site (sPY1) was not significantly phosphorylated in vitro, raising the possibility of a Src-induced tyrosine kinase cascade. The ability of Src to phosphorylate the EGF-R is consistent with the suggestion that the receptor can function as a kinase substrate independent of its intrinsic enzymatic activity, as implied by recent studies on signaling by kinase-defective EGF-Rs.