Abstract: Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser³³⁸, a crucial event for Raf-1 activation. Here we report that mutation of Lys³⁷⁵ to Met diminishes phosphorylation of Ser³³⁸ on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr³⁴⁰/Tyr³⁴¹ are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser³³⁸ phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser⁴⁷¹ to Ala) in the activation segment also abolishes Ser³³⁸ phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser³³⁸ is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser⁴⁴⁵ on B-Raf, a site equivalent to Raf-1 Ser³³⁸. Finally, our data reveal that Ser³³⁸ is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser³³⁸ phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser³³⁸ on Raf-1 is autophosphorylated in response to mitogens.