We investigated the interaction of the avian retrovirus ppl2 protein with viral RNA to assess its possible role in virion assembly. Using chemical modification techniques, we found that reagents specific for lysine or arginine residues inactivated the RNA-binding capacity of the protein. The binding of ppl2 to 60S viral RNA was also strongly affected by pH (pKapp of 5.5); the affinity for viral RNA decreased by as much as 40-fold after protonation of one or more titratable groups on the protein. When the protein was cleaved by cyanogen bromide, each of the two polypeptide products bound to RNA (with low affinity), but pH dependence was lost. Thus, an intact protein was required for this effect. Since histidine and phosphoserine residues have pKa values close to the pKapp of the ppl2-RNA interaction, they were studied to determine whether they were involved in this process. Each of the two histidyl residues in ppl2 had pKa values of 6.2, as determined by proton nuclear magnetic resonance titrations, values too high to account for the pKapp of binding. The involvement of phosphoserine residues, which have pKa values similar to the pKapp, was investigated by removal of phosphate from ppl2. When phosphate groups were chemically or enzymatically removed from the avian myeloblastosis virus, Rous sarcoma virus (Pr-C), and PR-E 95C virus ppl2 proteins, the Kapp for binding 60S viral RNA was reduced 100-fold at pH 7.5. Thus, it seems possible that phosphorylation of the ppl2 protein could favor viral nucleocapsid formation by increasing its affinity for the viral RNA genome. Dephosphorylation could provide for its release from the viral RNA during reverse transcription after viral infection of cells.