Linear EBV genomes undergo a transition to the circular form characteristic of latency by 16-20 h post-infection. This transition requires that the infected cells be activated to the G1 stage of the cell cycle. Cellular proliferation and expression of the activation marker CD23 were not required. Nevertheless, 36 h post-infection, only cells expressing CD23 contained covalently closed, circular episomes (CCC), at an average of one copy per cell. Since the presence of CD23 at this time is predictive that a cell will immortalize, we suggest that the presence of CCC is required for CD23 expression and subsequent immortalization. The one CCC present in each CD23+ cell did not undergo amplification until well after the cells had acquired all of the characteristic phenotypic markers of immortalization. Therefore, while amplification is not necessary for proliferation and immortalization, circularization of a single genome is crucial to the establishment and maintenance of latency by EBV.