This chapter describes new methods to study poliovirus assembly and the encapsidation of genomic RNA. Poliovirus, a member of the Picornaviridae family, is a small nonenveloped icosahedral virus that encapsidates a single genome molecule of the positive sense. The capsid shell that encloses the viral RNA genome is composed of 60 copies of each of four structural proteins: VP1 (33 kDa), VP2 (30 kDa), VP3 (26 kDa), and VP4 (7.5 kDa). The availability of an infectious clone for poliovirus has made it possible to use molecular genetics for dissecting the assembly and encapsidation process. This chapter describes the growth and characterization of the recombinant vaccinia viruses that express the poliovirus capsid precursor protein and the 3CD proteinase and the use of these recombinant vectors to study P1 cleavage and the assembly of subviral particles. The chapter discusses the use of the recombinant vaccinia virus expressing the P1 capsid precursor in conjunction with the defective poliovirus genome to study the individual steps of poliovirus assembly and encapsidation. The derivation of recombinant vaccinia viruses VV-P1, which expresses the poliovirus P1 capsid precursor protein, and VV-P3, which expresses the poliovirus 3CD proteinase, is described in the chapter. © 1995, Elsevier Inc.