Purified preparations of the Helminthosporium victoriae 190S (Hv190S) virus contain two sedimenting components that differ in capsid structure. The slower sedimenting component (190S-1) contained p88 and p83 as the major capsid proteins; the faster component (190S-2) contained p88 and p78. Previous peptide-mapping studies have shown the three capsid proteins to be closely related. Analysis by SDS-PAGE of in vivo-radiolabeled Hv190S virions indicated that 32P was predominantly incorporated in p88. Significantly less was detected in p83 and none in p78. Similar results were obtained in in vitro phosphorylation studies using [gamma-32P]ATP and purified 190S-1 and 190S-2. The in vitro results suggest that the Hv190S virions copurify with a protein kinase activity that catalyzes the transfer of gamma-phosphate from ATP to a target protein, presumably p78 in the 190S-2 virions and p83 in the 190S-1 component. Selective chemical cleavage at tryptophan residues of in vitro 32P-labeled capsid proteins revealed four labeled peptides among the cleavage products of both p83 and p88. Radioiodination studies with intact Hv190S virions indicated that p88 and p83, but not the nonphosphorylated p78, were accessible to iodination, suggesting that capsid protein phosphorylation entailed conformational changes.