E. coli RecA recombinase catalyzes the homology pairing and strand exchange reactions in homologous recombinational repair. RecA must compete with single-stranded DNA binding proteins (SSB) for single-stranded DNA (ssDNA) substrates to form RecA nucleoprotein filaments, as the first step of this repair process. It has been suggested that RecA filaments assemble mainly by binding and extending onto the free ssDNA region not covered by SSB, or are assisted by mediators. Using the tethered particle motion (TPM) technique, we monitored individual RecA filament assembly on SSB-wrapped ssDNA in real-time. Nucleation times of the RecA E38K nucleoprotein filament assembly showed no apparent dependence among DNA substrates with various ssDNA gap lengths (from 60 to 100 nucleotides) wrapped by one SSB in the (SSB)65 binding mode. Our data have shown an unexpected RecA filament assembly mechanism in which a RecA-SSB-ssDNA interaction exists. Four additional pieces of evidence support our claim: the nucleation times of the RecA assembly varied (1) when DNA substrates contained different numbers of bound SSB tetramers; (2) when the SSB wrapping mode conversion is induced; (3) when SSB C-terminus truncation mutants are used; and (4) when an excess of C-terminal peptide of SSB is present. Thus, a RecA-SSB interaction should be included in discussing RecA regulatory mechanism.