The existence of nonintegrated plasmid-chromosome complexes was deduced previously from the cosedimentation of covalently closed, circular plasmids with host folded chromosomes. It is now shown that about 70-90% of the covalently closed, circular F DNA could be released in vitro from chromosome complexes by RNase treatment, but not by protease, Sarkosyl or ethidium bromide. Consistent with the in vitro studies, E. coli cells treated for 5 min with rifampin, an inhibitor of RNA initiation, released upon lysis 90% of their plasmid DNA as freely sedimenting molecules.