Chromosomal common fragile sites (CFSs) are genetically unstable regions of the genome that are induced by conditions that impair DNA replication. In this report, we show that treatment with the DNA polymerase inhibitor, aphidicolin (APH), slows the replication rate throughout S phase. To investigate the unusual sensitivity of CFSs to APH-induced replication stress, we examined replication dynamics within a 50 kb region of the most frequently expressed CFS, FRA3B. We mapped four origins of replication, ori 1-4, using two independent methods. In untreated cells, we detected significantly less newly replicated DNA at FRA3B ori 1-3, as compared with three control origins located within non-fragile regions (NCFSs). In APH-treated cells, all FRA3B and control origins tested were active; however, there was a significant increase of nascent strand DNA at the control origins and, to a lesser extent, at the FRA3B ori 1-3. On the basis of these observations and the theoretical modeling of the nascent strand abundance assay developed in this study, we hypothesize that CFS origins may be less efficient, and that APH treatment slows replication fork movement near these origins to a greater extent, resulting in impaired DNA replication and, ultimately, leading to the genetic instability characteristic of CFSs.