A number of recent studies have reported that in papovaviruses such as simian virus 40 (SV40) and polyomavirus, the replication of the viral DNA in vivo is activated by the viral transcriptional enhancer or promoter sequences. Both viral and cellular transcriptional enhancers are well known for their ability to activate transcription in a position- and orientation-independent manner. In the present study, we investigated the effect of the position and orientation of the SV40 72-base-pair (bp) repeat enhancer on its replication activation function. We constructed plasmids containing one copy each of the SV40 core origin and enhancer placed in either order and orientation and at different distances from each other. We assayed the replication efficiencies of these plasmids in the presence of an internal control plasmid in COS-1 monkey kidney cells producing the SV40 T antigen required for replication. We found that the 72-bp repeat was capable of activating replication equally well in either orientation when placed 8 or 9 bp from the core origin. The activation of replication was totally abolished, and replication efficiencies in most instances were found to be lower than that obtained with the core origin alone, when the 72-bp repeat was separated from the core origin by distances of 99 bp or more. This was in direct contrast to the situation with polyomavirus, in which activation of replication by the homologous enhancer or by the SV40 72-bp repeat enhancer is known to be position independent. We also found that when the SV40 core origin and the 72-bp repeat enhancer were adjacent to each other, efficient activation of replication was obtained only if the end of the core origin containing the 17-bp A + T block was linked with the enhancer. In the other orientation of the core origin, activation of replication was either diminished or abolished. Hypotheses such as alteration of chromatin structure by the enhancer and interaction between trans-acting factors binding to the enhancer and the core origin mediating the activation effect are discussed.