The efficiency of single-stranded (ss) oligonucleotides binding at the secondary site of the RecA protein filament is demonstrated to depend on the strandedness of DNA bound at the primary site. When the primary site is occupied by a ss-oligonucleotide, the binding of another ss-oligonucleotide at the secondary site is characterized by higher affinity and a lower rate of dissociation than is the case when the primary site is occupied by a double-stranded oligonucleotide. In contrast to a DNA strand exchange reaction suppressed by a heterologous oligonucleotide bound at the secondary site of the RecA filament, the occupation of the secondary site by a heterologous oligonucleotide does not prevent renaturation between the oligonucleotides bound at the primary site and complementary oligonucleotides from solution demonstrating that the binding of a DNA strand in the secondary site is not a necessary intermediate step in RecA-promoted DNA renaturation.