Initiation of polyoma virus DNA replication is dependent on the activity of the early protein affected by the tsa mutations (large-T antigen). An in vitro DNA synthesizing system blocked at the initiation stage was designed by preparing nuclei from cells shifted to high temperature after infection with a polyoma tsa mutant. Addition to these nuclei of extracts from wild type virus-infected cells resulted in a limited, but reproducible stimulation of deoxynucleoside monophosphate incorporation. At least for a significant part, this stimulation was shown to correspond to an increased synthesis of molecules identified as polyoma replicative intermediates by their sedimentation coefficient and endonuclease Hpa II cleavage pattern. The non-random distribution of label observed among restriction fragments was that expected from an initiation event occuring at the physiological origin. This activity was reduced to background level in extracts from tsa-infected cells shifted to high temperature and was specifically inhibited by addition of Fab fragments from anti-polyoma virus T antigen immunoglobulins.