A novel molecular mechanism of autophosphorylation-dependent activation of the ser/thr S6/H4 kinase isolated from human placenta is described. Phosphopeptide mapping of the enzyme was used to determine the rate and extent of site-specific autophosphorylation. These data were correlated to phosphotransferase activity of the protein kinase. The results indicated that a sequential phosphorylation of two sites in the catalytic domain is required for maximum activation. Kinetic analysis determined that site 1 is modified by an intramolecular phosphorylation, and site 2 is modified by an intermolecular phosphorylation. On the basis of these data a model is proposed in which autophosphorylation of the pseudosubstrate domain and on a serine residue in subdomain VIII are both required for maximum activation of the S6/H4 kinase.