The structure of harlequin-stained chromosomes following substitution with low levels of 5-bromodeoxyuridine (BrdUrd) over two cell cycles and high levels over the last part of one cycle (replication banding) was studied in Chinese hamster ovary (CHO) cells. By using correlative light (LM) and scanning electron microscopy (SEM), it was shown that the effects of both the ultraviolet light (u.v.) and hot SSC treatment steps of the harlequin staining procedure were necessary to obtain sister-chromatid differentiation (SCD) or replication banding. u.v. treatment alone resulted in dark Giemsa staining of both chromatids with SEM morphology of short compact protuberances and an overall flattened smooth appearance in both the unsubstituted and BrdUrd-substituted chromatids, a morphology essentially similar to that of untreated chromosomes. SSC alone on the other hand resulted in dark-staining chromatids with an SEM morphology of raised, loosely packed loops of fibres in both types of chromatids. u.v. and SSC treatment together resulted in differentiation, with dark-staining unifilarly (TB) chromatids in the LM corresponding to raised loosely packed loops in the SEM and pale bifilarly (BB) chromatids corresponding to the smooth compact flattened SEM appearance. Where the BrdUrd-substituted strand became the template (BT), or when the nascent strand TB contained high levels of BrdUrd substitution in replication banding, the chromatid stained pale and showed the compact smooth appearance in the SEM. The Giemsa staining ability and ultrastructural morphology of harlequin staining is discussed with respect to putative DNA loss and also in terms of preferential protein-protein, protein-DNA cross-linkage in BrdUrd-containing DNA. These changes are also compared with the ultrastructural morphology observed after other banding methods, where deterioration of protein and DNA-protein interaction resulting in aggregation of chromatin fibres appears to be the major mechanism.