The Saccharomyces yeast plasmid, 2-micron circle, encodes a partitioning system that ensures equidistribution of plasmid molecules to both progeny following cell division. This system consists of two proteins encoded in plasmid genes REP1 and REP2 and a cis-active noncoding locus, designated REP3. We have raised antibodies against a REP1 beta-galactosidase fusion protein and used them to identify the authentic REP1 protein in plasmid-bearing yeast cells. We find that REP1 protein is located exclusively in the nucleus and co-purifies with a karyoskeletal protein subfraction operationally and morphologically equivalent to the nuclear matrix-pore complex-lamina fraction of higher cells. The carboxyl half of the REP1 protein exhibits strong sequence homology to myosin heavy chain, vimentin, and nuclear lamins A and C, indicating a fibrous structure for the protein. From these observations, we suggest that REP1 protein may promote plasmid partitioning by intercalating into the nuclear lamina of the host cell to provide dispersed anchorage sites for attachment of plasmid molecules.