The kinetic mechanism of transfer of the homotetrameric Escherichia coli SSB protein between ssDNA molecules was studied using stopped-flow experiments. Dissociation of SSB from the donor ssDNA was monitored after addition of a large excess of unlabeled acceptor ssDNA by using either SSB tryptophan fluorescence or the fluorescence of a ssDNA labeled with an extrinsic fluorophore [fluorescein (F) or Cy3]. The dominant pathway for SSB dissociation occurs by a "direct transfer" mechanism in which an intermediate composed of two DNA molecules bound to one SSB tetramer forms transiently prior to the release of the acceptor DNA. When an initial 1:1 SSB-ssDNA complex is formed with (dT)70 in the fully wrapped (SSB)65 mode so that all four SSB subunits are bound to (dT)70, the formation of the ternary intermediate complex occurs slowly with an apparent bimolecular rate constant, k2,app, ranging from 1.2 × 103 M-1 s-1 (0.2 M NaCl) to ∼5.1 × 103 M-1 s-1 (0.4 M NaBr), and this rate limits the overall rate of the transfer reaction (pH 8.1, 25°C). These rate constants are ∼7 × 105- and ∼7 × 104-fold lower, respectively, than those measured for binding of the same ssDNA to an unligated SSB tetramer to form a singly ligated complex. However, when an initial SSB-ssDNA complex is formed with (dT)35 so that only two SSB subunits interact with the DNA in an (SSB)35 complex, the formation of the ternary intermediate occurs much faster with a k2,app ranging from >6.3 × 107 M-1 s-1 (0.2 M NaCl) to 2.6 × 107 M-1 s-1 (0.4 M NaBr). For these experiments, the rate of dissociation of the donor ssDNA determines the overall rate of the transfer reaction. Hence, an SSB tetramer can be transferred from one ssDNA molecule to another without proceeding through a free protein intermediate, and the rate of transfer is determined by the availability of free DNA binding sites within the initial SSB-ssDNA donor complex. Such a mechanism may be used to recycle SSB tetramers between old and newly formed ssDNA regions during lagging strand DNA replication.