The chromosome of R6K contains multiple origins of replication. The origin gamma is infrequently used in the original plasmid and remains "silent" in certain miniplasmid derivatives. The inactivation of the origin is caused by a natural origin silencer located adjacent to the minimal ori gamma sequence. The silencer functions in cis and has no trans activity. It has functional polarity and works only in one orientation when present immediately downstream from ori gamma. The silencer apparently initiates an RNA that invades ori gamma and turns it off either by competing with a primer RNA or by disrupting ori gamma structure. As predicted, removal of the silencer blocks the synthesis of silencer RNA and derepresses the origin.