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Extracts from human adenovirus type 5 (Ad5)-transformed cells were fractionated by ammonium sulphate precipitation, DEAE-Sephacel chromatography and glycerol gradient centrifugation. In all cases, protein kinase activity co-purified with the 58000 mol. wt. polypeptide (58K) coded for by the early 1B region of Ad5. Kinase activity was also precipitated by an antiserum raised against a synthetic peptide corresponding to the predicted carboxy terminus of 58K. These data suggest that protein kinase activity is associated with 58K, either intrinsically or in an enzyme bound to 58K.