In a subclone derived from mouse L(tk-) cells, we found a plasmid present in a high copy number (greater than 5000 copies per cell) that was stably maintained extrachromosomally without any cytopathic effect to the host cells. This plasmid, termed L factor, has two forms: 5.3 and 5.5 kilobase pairs. DNA sequencing and restriction enzyme mapping showed that, although the structure contains DNA sequences common to polyoma virus, plasmid sequences belonging to the regulatory region (the enhancer region) and other regions are quite different from those in polyoma. In cells bearing the plasmid, we detected a low level of material that cross-reacts with antibody to polyoma tumor antigens, suggesting that the plasmids replicate and are maintained in the cells by a mechanism different from that functioning during propagation following infection of papovaviruses.