DNA molecules with restricted binding of intercalating dyes are observed as replicative intermediates during the replication of bacteriophage M-13 duplex DNA in a cellular system in vitro prepared by plasmolysis of M-13-am5-infected Escherichia coli cells. Restriction of dye binding is abolished by heating the DNA to 80 degrees C, but can be recovered by slow cooling of the heat-treated DNA. Radioactive pulse-label incorporated by these molecules is found exclusively in elongated viral strands of more than one genome length. In the electron microscope this DNA fraction is seen to contain a significant number of duplex DNA rings with two single-stranded tails protruding from the same region of the ring. It is proposed that these structures arise by branch migration during the isolation of replicating molecules containing only one single-stranded tail. The topological constraint in these molecules is most likely caused by base-pairing between partially complementary regions of the two single-stranded tails.