The replication of eukaryotic chromosomes takes place throughout S phase, but little is known how this process is organized in space and time. Early and late replicating chromosomal domains appear to localize to distinct spatial compartments of the nucleus where DNA synthesis can take place at defined times during S phase. In general, transcriptionally active chromatin replicates early in S phase whereas transcriptionally inactive chromatin replicates later. Here we provide evidence for significant deviation from this dogma in mouse NIH3T3 cells. While the bulk pericentromeric heterochromatin replicates exclusively during mid to late S phase, centromeric DNA domains associated with constitutive kinetochore proteins are replicated throughout all stages of S phase. On an average, 12+/-4% of centromeres replicate in early S phase. Early replication of a subset of centromeres was also detected in living C2C12 murine cells. Thus, in contrast to expectation, late replication is not an obligatory feature of centromeric heterochromatin in murine cells and it does not determine their 'heterochromatic state'.