High-resolution replication banding was performed to (1) delineate at the prometaphase level small interstitial deletions of chromosome 13 in two unrelated patients with retinoblastoma (Rb) and (2) study the influence of these deletions in the replication autonomy of the regions involved. The present study indicates that, in spite of the fact that break points separate (split) bands ql4 and q21 in different segments, these deletions do not produce a position effect or alteration in the replication behavior of the abnormal chromosome 13’s. Each segment in the deleted chromosomes replicates with a chronology and pattern identical to the normal homolog’s. The present data also indicate that metaphase bands are not only autonomous foci of replication but that they can be further divided into subunits of replication which correspond to sub-bands observed in more elongated prometaphase chromosomes. Our results strongly suggest that prometaphase sub-bands represent units of chromatid organization and replicat