Abstract The interaction of the Escherichia coli single strand binding (SSB) protein with single-stranded DNA is complex, since a number of different binding modes have been observed, with different DNA site sizes and binding properties and the transitions among these binding modes are strongly influenced by solution conditions in vitro. Recent experiments have suggested the existence of negative co-operativity among the multiple DNA binding sites within individual SSB tetramers. In order to probe this negative co-operativity, we have examined the binding of a series of oligonucleotides of varying length, using the quenching of the intrinsic SSB protein fluorescence to monitor binding. The stoichiometries for saturation of the SSB tetramer are 4, 2, 2, 1 and 1, for the oligonucleotides, dT(pT) N−1 , with N = 16, 28, 35, 56 and 70, respectively, indicating that one molecule of either dT(pT) 27 or dT(pT) 34 interacts with two SSB subunits, whereas one molecule of dT(pT) 15 interacts with only a single subunit. Saturation of the SSB tetramer with dT(pT) 15, dT(pT) 34, dT(pT) 69 or poly(dT) results in 85 to 90% quenching of the SSB fluorescence, whereas saturation with dT(pT) 27 or dT(pT) 55 results in only 80% and 72% quenching, respectively. Therefore, a single-stranded DNA of at least 64 nucleotides is required to wrap around an SSB tetramer fully and interact with all four subunits. A quenching of 50(± 2)% is observed upon filling only half of the subunits with either one molecule of dT(pT) 34 or two molecules of dT(pT) 15, which agrees with the quenching and site size observed in the (SSB) 35 polynucleotide binding mode. Direct binding measurements indicate that the binding of dT(pT) 27 to its second site is influenced by the oligonucleotide that occupies the first binding site (either dT(pT) 27 or dT(pT) 34), providing proof for the existence of a true negative co-operativity. This negative co-operativity is observed also for the binding of the shorter oligonucleotide, dT(pT) 15. A statistical thermodynamic (“square”) model gives an excellent description of the binding of all oligonucleotides possessing multiple sites on the SSB tetramer, based on only two interaction constants, the intrinsic binding constant, K N , and the negative co-operativity parameter, σ N. These data indicate that the binding sites (subunits) on the unliganded SSB tetramer are all equivalent, but that a non-equivalence between dimers of subunits within the tetramer is induced upon binding ssDNA.