Phorbol esters, such as phorbol myristate acetate (PMA), cause differentiation of U937 human monomyelocytic cells along the macrophage pathway. Within 15 min of PMA treatment DNA binding of the c-jun transcription factor is increased and is accompanied by rapid changes in the phosphate content of the c-jun protein. Phorbol esters stimulate phosphorylation of serines 63 and 73 located within the A1 transactivation domain of c-Jun that have previously been shown to positively regulate activity. A protein kinase activity is detectable in extracts of phorbol ester-treated U937 cells that specifically targets these two serines. Using novel assays, the protein kinase activity has been purified over 1000-fold. The major portion of protein kinase activity co-chromatographs over three columns with pp42/44 mitogen-activated protein kinases as judged by immunological methods. The significance of these results with respect to mitogen-induced transcription of AP-1-responsive genes is discussed.