Highly purified African swine fever virus contains a cyclic AMP-independent protein kinase which phosphorylates endogenous virus proteins with a specific activity of about 0.45 pmol/microgram of virus protein. The major substrates for the virion protein kinase in vitro were the structural proteins p10 and p9. Both proteins were phosphorylated preferentially at serine residues. A possible relationship between protein p10 phosphorylation and RNA synthesis in vitro by the virion-associated RNA polymerase is suggested by the finding that N-alpha-tosyl-L-lysyl-chloromethyl ketone inhibited both phosphorylation of p10 and transcription. Two phosphoproteins, with molecular masses of 35 and 17 kDa, were found in African swine fever virus purified from infected Vero cells labeled with [32P]phosphate. A phosphopolypeptide with a molecular mass of about 35 kDa was found in the cytoplasm of infected Vero cells.