The FRA16D, at 16q23, spans the WWOX gene and is one of the most highly expressed common fragile sites observed when DNA replication is perturbed by aphidicolin. Several lines of evidence suggest that fragile sites are regions of DNA that are unusually sensitive to interference during replication. We have determined that the FRA16D alleles replicate in a synchronous fashion and that replication of these sequences occurs primarily in late S phase extending into G2 phase. Exposure to aphidicolin, an inhibitor of DNA polymerase alpha, results in a modest increase in cells with replication of FRA16D sequences in early S phase. This may represent initiation of replication in early S phase coupled with slow replication progression, or, alternatively, these cells may have passed through mitosis, entered the G1-S phase of the next cell cycle, and initiated replication/repair. Our results support a model in which common fragile sites are sequences that may initiate replication in early-mid S phase but are slow to complete replication, and the chromosomal breaks and gaps observed in metaphase cells result from unreplicated DNA.