DNA was isolated from cells of Saccharomyces cerevisiae incubated under conditions that enriched for DNA replication intermediates. A novel form of the 2 microns plasmid was detected, in which two monomeric or dimeric circles were joined by a linear double-stranded segment of variable length. We suggest that this molecule is a consequence of site-specific recombination within a dimeric DNA molecule during DNA replication. The existence of this molecule provides supporting physical evidence for a variant of the model of 2 mu plasmid amplification first proposed by Futcher (1986).