The co-operative nature of the binding of the Escherichia coli single strand binding protein (SSB) to single-stranded nucleic acids has been examined over a range of salt concentrations (NaCl and MgCl2) to determine if different degrees of binding co-operativity are associated with the two SSB binding modes that have been identified recently. Quantitative estimates of the binding properties, including the co-operativity parameter, (ω, of SSB to single-stranded DNA and RNA homopolynucleotides have been obtained from equilibrium binding isotherms, at high salt (≥0.2 m-NaCl), by monitoring the fluorescence quenching of the SSB upon binding. Under these high salt conditions, where only the high site size SSB binding mode exists (65 ± 5 nucleotides per tetramer), we find only moderate co-operativity for SSB binding to both DNA and RNA, (ω = 50 ± 10), independent of the concentration of salt. This value for ω is much lower than most previous estimates. At lower concentrations of NaCl, where the low site size SSB binding mode (33 ± 3 nucleotides/tetramer) exists, but where SSB affinity for single-stranded DNA is too high to estimate co-operativity from classical binding isotherms, we have used an agarose gel electrophoresis technique to qualitatively examine SSB co-operativity with single-stranded (ss) M13 phage DNA. The apparent binding co-operativity increases dramatically below 0.20 m-NaCl, as judged by the extremely non-random distribution of SSB among the ssM13 DNA population at low SSB to DNA ratios. However, the highly co-operative complexes are not at equilibrium at low SSB/DNA binding densities, but are formed only transiently when SSB and ssDNA are directly mixed at low concentrations of NaCl. The conversions of these metastable, highly co-operative SSB-ssDNA complexes to their equilibrium, low co-operativity form is very slow at low concentrations of NaCl. At equilibrium, the SSB-ssDNA complexes seem to possess the same low degree of co-operativity (ω = 50 ± 10) under all conditions tested. However, the highly co-operative mode of SSB binding, although metastable, may be important during non-equilibrium processes such as DNA replication. The possible relation between the two SSB binding modes, which differ in site size by a factor of two, and the high and low co-operativity complexes, which we report here, is discussed. The different SSB-ssDNA complexes that are observed to form in vitro may each function selectively in replication, recombination and repair processes in vivo.