Chinese hamster ovary (CHO) cells, which are arrested at the G1/S-phase of the cell cycle with hydroxyurea, enter mitosis prematurely when treated with caffeine [Schlegel and Pardee, 1986; Science 233-1264-1266]. Such mitotic cells with unreplicated genomes (MUGs) can assemble a mitotic spindle and progress through M-phase even in the absence of intact, replicated chromosomes [Brinkley et al., 1988: Nature 336:251-254; Zinkowski et al., 1991: J. Cell Biol. 113:1091-1110; Christy et al., 1995: Protoplasma 186:193-200]. In order to better define the role of the spindle in chromosome movement, we compared the structure and assembly of mitotic spindles and analyzed the nature of kinetochore association and movement in control cells and MUGs. The mitotic spindles in MUGs display the same morphological features and dynamic properties of assembly-disassembly as seen in normal spindles. Although multiple centromere-kinetochore fragments (CKFs), derived from fragmented chromosomes, interact with and attach to spindle microtubules in both orthodox and unorthodox ways, they nevertheless become aligned on the metaphase plate. Prometaphase congression and alignment at metaphase is achieved in MUGs even though CKFs represent kinetochore fragments that originate from unreplicated chromosomes and, therefore, lack "sister kinetochore" orientation such as seen in chromosomes of control cells. Our study supports the notion that much of the "information" needed for prometaphase chromosome movement and alignment is endemic to the spindle.