Topoisomerase IIα (topoIIα) and 13S condensin are both required for mitotic chromosome assembly. Here we show that they constitute the two main components of the chromosomal scaffold on histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. TopoIIα and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topoIIα and condensin generates a barber pole appearance of the scaffolding, where topoIIα- and condensin-enriched “beads” alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatids; they lack this complex but contain a topoIIα-defined (-mediated?) scaffolding. Condensin associates only during the pro- to metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding.