The poliovirus capsid precursor polyprotein, P1, is cotranslationally modified by the addition of myristic acid. We have examined the importance of myristylation of the P1 capsid precursor during the poliovirus assembly process by using a recently described recombinant vaccinia virus expression system which allows the independent production of the poliovirus P1 protein and the poliovirus 3CD proteinase (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). We constructed a site-directed mutation in the poliovirus cDNA encoding an alanine at the second amino acid position of P1 in place of the glycine residue required for the myristic acid addition and isolated a recombinant vaccinia virus (VVP1myr-) that expressed a nonmyristylated form of the P1 capsid precursor. The 3CD proteinase expressed by a coinfecting vaccinia virus, VVP3, proteolytically processed the nonmyristylated precursor P1 expressed by VVP1myr-. However, the processed capsid proteins, VP0, VP3, and VP1, did not assemble into 14S or 75S subviral particles, in contrast to the VP0, VP3, and VP1 proteins derived from the myristylated P1 precursor. When cells were coinfected with VVP1myr- and poliovirus type 1, the nonmyristylated P1 precursor expressed by VVP1myr- was processed by 3CD expressed by poliovirus, and the nonmyristylated VP0-VP3-VP1 (VP0-3-1) protomers were incorporated into capsid particles and virions which sedimented through a 30% sucrose cushion. Thus, the nonmyristylated P1 precursor and VP0-3-1 protomers were not excluded from sites of virion assembly, and the assembly defects observed for the nonmyristylated protomers were overcome in the presence of myristylated capsid protomers expressed by poliovirus. We conclude that myristylation of the poliovirus P1 capsid precursor plays an important role during poliovirus assembly by facilitating the appropriate interactions required between 5S protomer subunits to form stable 14S pentamers. The results of these studies demonstrate that the independent expression of the poliovirus P1 and 3CD proteins by using recombinant vaccinia viruses provides a unique experimental tool for analyzing the dynamics of the poliovirus assembly process.