Escherichia coli SSB tetramers can bind to single stranded (ss) DNA in several binding modes. At 25 degrees C, pH 8.1, SSB can form at least three distinct binding modes, (SSB)n, where the number of nucleotides occluded per tetramer (n), can have values of 35, 56 or 65. Stability of the different modes is modulated by solution conditions, primarily the salt concentration and type, as well as the free SSB concentration. At least two different types of positive co-operative binding of SSB to ssDNA have also been observed, which appear to be correlated with different SSB binding modes. The (SSB)65 mode, which dominates at monovalent salt concentrations > 0.2 M, displays only moderate, "limited" co-operative binding in which clustering of SSB is limited to the formation of dimers of tetramers (octamers). However, at lower salt concentrations, "unlimited" co-operative binding is observed in which long SSB clusters can form, similar to the behavior observed for the phage T4 gene 32 protein. It has been proposed that unlimited co-operativity is linked to the (SSB)35 binding mode; however, this has not been verified since quantitative estimates of the co-operativity in this binding mode are difficult on long ssDNA. To estimate the nearest-neighbor co-operativity parameter in the (SSB)35 mode, we have examined the equilibrium binding of SSB to the oligodeoxynucleotide, dA(pA)69. Under certain conditions, 1:1 complexes, in which all four SSB subunits interact with the dA(pA)69, can form at low SSB binding densities, whereas 2:1 complexes, in which both SSB tetramers bind to DNA using only two subunits, can form at high SSB binding densities. These 2:1 complexes serve as a model for co-operative binding in the (SSB)35 binding mode. We show that SSB tetramers bind in this mode with a minimum nearest-neighbor co-operativity parameter of omega 35 = 1.0 x 10(5) (0.125 M NaCl, pH 8.1, 25 degrees C). This indicates that the nearest-neighbor co-operativities for SSB tetramers bound to ssDNA in the (SSB)35 versus the (SSB)65 mode differ qualitatively and quantitatively and suggests that the (SSB)35 mode is responsible for the ability of SSB protein to form long clusters on ssDNA. If the ability of helix destabilizing proteins to form uninterrupted protein clusters on ssDNA is important in DNA replication, then it is likely that SSB uses its (SSB)35 mode to function in this capacity.