Ten protein kinases have been assayed for their ability to phosphorylate in vitro the recombinant bovine PrP (25-242) (rbPrP). Substantial phosphorylation was observed with PKC, CK2, and two tyrosine kinases, Lyn and c-Fgr. With regard to CK2, phosphorylation occurs at Ser 154 with a stoichiometry of about 0.1 mol phosphate/mol rbPrP, which is doubled by mild heat treatment of rbPrP. Heat also reduces the overall protein ellipticity, suggesting that reversibly unfolded conformers are more susceptible to phosphorylation. Our data disclose the possibility that phosphorylation might modulate PrP biological activity.