Numerous studies have demonstrated that DNA replication initiates within the 30 kB non-transcribed spacer (NTS) region of the human ribosomal RNA gene (rDNA). Using a series of closely spaced primer pairs to measure nascent leading strand abundance in mid and late S phase cells isolated by centrifugal elutriation, we find evidence for one highly preferred initiation site and two less utilized sites within a 6 kb region of the NTS. The initiation sites colocalize with significant DNA unwinding elements (DUEs), matrix attachment regions (MARs), and ARS-like sequences. An intrinsic DNA bending site was localized by circular permutation analysis to within several hundred base pairs of one initiation site. While DUE and MAR elements occur elsewhere throughout the 43 kb rDNA sequence, the close association of DUE and MAR elements occurs only near replication initiation sites, a juxtaposition also seen in other well-studied mammalian replication initiation sites. The utilization of rDNA initiation sites close to DUE and MAR elements in mid and late S phase, but not in very early S phase as previously shown, suggests that in rRNA genes, contributions from these sequence-associated properties may be more significant to initiation sites associated with transcriptionally inactive genes, than to initiation sites associated with transcriptionally active genes.