Human myeloid leukemia cells respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and other activators of protein kinase C (PKC) with induction of monocytic differentiation. The present studies demonstrated that treatment of U-937 and HL-60 myeloid leukemia cells with TPA, phorbol-12,13-dibutyrate, or bryostatin 1 was associated with the induction of stress-activated protein kinase (SAPK). In contrast, TPA-resistant TUR and HL-525 cell variants deficient in PKCβ failed to respond to activators of PKC with the induction of SAPK. A direct role for PKCβ in TPA-induced SAPK activity in TUR and HL-525 cells that stably express PKCβ was confirmed. We showed that TPA induced the association of PKCβ with MEK kinase 1 (MEKK-1), an upstream effector of the SAPK/ERK kinase 1 (SEK1)→SAPK cascade. The results also demonstrated that PKCβ phosphorylated and activated MEKK-1 in vitro. The functional role of MEKK-1 in TPA-induced SAPK activity was further supported by the demonstration that the expression of a dominant negative MEKK-1 mutant abrogated this response. These findings indicate that PKCβ activation is necessary for activation of the MEKK-1→SEK1→SAPK cascade in the TPA response of myeloid leukemia cells.