The SeV P protein is found as a homotrimer (P3) when it is expressed in mammalian cells, and trimerization is mediated by a predicted coiled-coil motif which maps within amino acids (aa) 344 to 411 (the BoxA region). The bacterially expressed protein also appears to be trimeric, apparently precluding a role for phosphorylation in the association of the P monomers. I have examined the role of P trimerization both in the protein's interaction with the nucleocapsid (N:RNA) template and in the protein's function on the template during RNA synthesis. As with the results of earlier experiments (32), I found that both the BoxA and BoxC (aa 479 to 568) regions were required for stable binding of P to the N:RNA. Binding was also observed with P proteins containing less than three BoxC regions, suggesting that trimerization may be required to permit contacts between multiple BoxC regions and the N:RNA. However, these heterologous trimers failed to function in viral RNA synthesis, indicating that the third C-terminal leg of the trimer plays an essential role in P function on the template. We speculate that this function may involve the movement of P (and possibly the polymerase complex) on the template and the maintenance of processivity.