Replication of poliovirus (PV) genomic RNA in HeLa cells has previously been found to start at distinct sites at the nuclear periphery. In the present study, the earliest steps in the virus replication cycle, i.e. the appearance and intracellular translocation of viral protein and negative-strand RNA prior to positive-strand RNA synthesis, were followed. During translation, positive-strand RNA and newly synthesized viral protein presented as a dispersed endoplasmic reticulum (ER)-like pattern. Concomitant with translation, individual PV vesicle clusters emerged at the ER and formed nascent replication complexes, which contained newly synthesized negative-strand RNA. The complexes rapidly moved centripetally, in a microtubule-dependent way, to the perinuclear area to engage in positive-strand viral RNA synthesis. Replication complexes made transcriptionally silent with guanidine/HCl followed the anterograde membrane pathway to the Golgi complex within the microtubule-organizing centre (MTOC), whereas replication complexes active in positive-strand RNA synthesis were retained at the nuclear periphery. If the silent replication complexes that had accumulated at the MTOC were released from the guanidine block, transcription was not readily resumed. Rather, positive-strand RNA was redistributed back to the ER to start, after a lag phase, translation, followed by negative- and positive-strand RNA synthesis in replication complexes migrating to the nuclear periphery. As some of the findings appear to be in contrast to events reported in cell-free guanidine-synchronized translation/transcription systems, implications for the comparison of in vitro systems with the living cell are discussed.