Abstract Replication initiation events are suppressed over the SV40 core origin in vitro;they are also greatly reduced over sequences flanking the origin which contain binding sites for several transcription factors. To address the biochemical basis for the gap in initiation events over the flanking sequences, initial synthesis events have been characterized on templates lacking these sequences. Herein, it is demonstrated that previously functional initiation sites are nearly inactive when moved to positions that are proximal to the core origin. Thus, the gap in initiation events depends, in part, on the proximity of the initiation sites to the SV40 core origin. Additional experiments demonstrate that removal of the flanking sequences had little or no effect on DNA unwinding or on the efficiency of initiation of DNA synthesis in vitro.These results indicate that, under our in vitroconditions, initiation of SV40 DNA synthesis is not enhanced by binding of transcription factors to the flanking sequences.