pSM19035-encoded omega protein forms a dimer (omega2) that binds to a set of 7-bp repeats with sequence 5'-NATCACN-3'. Upon binding to its cognate sites, omega2 regulates transcription of genes required for copy number control and stable inheritance of plasmids, and promotes accurate plasmid segregation. Protein omega2 binds poorly to one heptad but the affinity to DNA increases with two and more unspaced heptads in direct or inverted orientation. DNA titration of increasing numbers of heptads with omega2, monitored by circular dichroism measurements, indicates the binding of one omega2 to one heptad (omega2:heptad stoichiometry of 1:1). Spacing of two directly or inversely oriented heptads by 1 to 7 bp reduces the affinity of the protein for its cognate target site. The binding affinity of omega2 for two directly repeated heptads was severely reduced if one of the base pairs of the core 5'-ATCAC-3' sequence of one of the heptads was individually substituted by any other base pair. Hydroxyl radical footprinting shows a protection pattern at the 5'-ATCAC-3' core. These data suggest that each heptad defines an operator half-site and that tight binding of the symmetric omega2 to the central 5'-TCA-3' core of symmetric or asymmetric targets (differently oriented heptads) is probably achieved by structural changes of DNA and/or protein or both.