The functional organization of the eukaryotic chromosome was first elucidated at a molecular level in the budding yeast, Saccharomyces cerevisiae , providing the basis for the successful creation of yeast artificial chromosomes (YACs) (1 ). The structures that confer chromosome function have been far more difficult to determine in multicellular eukaryotes, both because of their greater complexity and size. Over the last decade, various strategies have been developed for creating engineered human/mammalian chromosomes. These fall into two broad categories: the use of naked DNA containing sequences capable of de novo chromosome formation (the “bottom-up” approach) or the manipulation and modification of existing chromosomes (the “top-down” approach). In this review, we will refer to chromosomes formed from naked input DNA as artificial chromosomes (ACs) and those produced from existing chromosomes as mini- or derivative-chromosomes (depending on their final size).