Yeast artificial chromosomes (YACs) containing or lacking a biochemically defined DNA replication origin were transferred from yeast to mammalian cells in order to determine whether origin-dependent autonomous replication would occur. A specialized YAC vector was designed to enable selection for YACs in mammalian cells and for monitoring YAC abundance in individual mammalian cells. All of eight clones made with linear and circularized YACs lacking the origin and seven of nine clones made with linear and circularized YACs containing the origin region contained single copies of the transfected YAC, along with various amounts of yeast DNA, integrated into single but different chromosomal sites. By contrast, two transformants derived from circularized YACs containing the putative replication origin showed very heterogeneous YAC copy number and numerous integration sites when analyzed after many generations of in vitro propagation. Analysis of both clones at an early time after fusion revealed variously sized extrachromosomal YAC/yeast structures reminiscent of the extrachromosomal elements found in some cells harboring amplified genes. The data are consistent with the interpretation that YACs containing a biochemically defined origin of replication can initially replicate autonomously, followed by integration into multiple chromosomal locations, as has been reported to occur in many examples of gene amplification in mammalian cells.