Typical of many transcriptional regulatory proteins, the lambdoid bacteriophage repressors bind cooperatively to multiple sites on DNA. This cooperative binding is essential for establishment and maintenance of phage lysogeny. In the phage, two repressor homodimers, one bound at each of the adjacent operator sites, interact to form the tetramer that is necessary for the cooperative binding of the repressor. Bacteriophage 434 repressor does not form tetramers in the absence of DNA, and the mechanism by which the tetramer assembles on the two adjacent sites is unknown. Hence DNA binding may stimulate the repressor to form tetramers and formation of a repressor oligomer (> or = 3 monomers) on a single DNA sites may precede multisite binding. Consistent with these ideas, a complex containing three repressor molecules readily assembles on a single operator (O(R)1) site. Mutations that inhibit cooperative tetramer binding to the adjacent O(R)1 and O(R)2 sites also block formation of this complex. Together with other evidence, these findings show that the complex that forms on a single site assembles using the same interface as does the tetramer assembled on adjacent operator sites. Adding additional O(R)1 DNA dissociates the oligomeric repressor-DNA complexes into dimeric repressor-O(R)1 complexes. In contrast, adding O(R)2 to these complexes results in the formation of a repressor oligomer containing an O(R)2 and an O(R)1 site. The observation that a repressor oligomer bound to two O(R)1 sites is less stable than the one formed between repressor dimers bound to O(R)1 and O(R)2 implies that DNA allosterically influences the structure of the 434 repressor. Together these findings suggest that an O(R)1-bound repressor may cooperatively help repressor bind to O(R)2 by recruiting an additional repressor molecule from solution that subsequently occupies O(R)2.