Cellular processes involved in fragile site expression have been investigated by studying the effect on the replication pattern of the commonest fragile site FRA3B of RNA interference (RNAi)-mediated sister maintenance chromosome 1 (SMC1) inhibition in normal human fibroblasts. Replication timing of FRA3B in G2 was studied by bromodeoxyuridine (BrdU) labeling for the final 2h of cell culture whereas in the S phase was investigated by a fluorescence in situ hybridization (FISH)-based approach through the analysis of clones spanning the FRA3B region. Results showed that FRA3B is normally late replicated even though it is not expressed in untreated cells. On the other hand, SMC1 inhibition leads to FRA3B expression even if the percent of late replicated cells is comparable to control cells. These results obtained by analysing the commonest fragile site suggest that SMC1 plays a role in protecting late replicating regions from stresses occurring in the final steps of genome replication and that delayed replication is necessary but not sufficient for inducing fragile site expression.