Previous studies have demonstrated that the 21- or the 72-bp repeat transcriptional control elements enhance the efficiency of SV40 DNA replication in vivo, provided either of these repeats is located near the end of the core replication origin containing the 17-bp A + T-containing sequence. Using two sets of point mutants we have investigated the contributions of the various sequence motifs present in the 21- or the 72-bp repeats toward activation of replication. Regarding the contribution of the six GC motif components of the 21-bp repeats, we find that GC motif I, located closest to the core origin, is dispensable for activation of replication. A mutation in GC-I in fact causes an increase in replication efficiency. We also find that GC motifs I and II present in the nontandem copy of the 21-bp repeats are not sufficient to activate replication. Our present study indicates that a combination of three GC motifs such as II, III, and IV (including one of the two perfect, tandem copies of the 21-bp repeats) is important for activation of replication. Regarding the 72-bp repeat transcriptional enhancer region, we find mutations in a number of its individual motifs to have a negative consequence on replication, with mutations in the GT-I*/TC-II and Sph-II/octamer motifs exhibiting the most negative effects. Overall, we find that the replication activation effects of the 21- and the 72-bp repeats require the participation of multiple motifs present in them. Cellular factors binding to these motifs are expected to mediate their replication activation effects. For the most part, the motifs required for activation of replication are the same as those reported in earlier studies to be important for efficient early and late viral mRNA transcription.