RecBCD is an ATP-dependent helicase and exonuclease, which generates 3'-single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. Critical to this function is its encounter with an regulatory DNA octamer called the Chi sequence, 5'-GCTGGTGG-3', which is known to stimulate recombination by the RecBCD pathway. RecBCD undergoes an unexplained modification following Chi encounter that results in attenuation of its 3'-5'exonuclease activity. There are several aspects of the Chi-RecBCD interactions that are still not understood, and most studies addressing these issues have done so by employing Chi- bearing duplexes in various biochemical assays for RecBCD functions. The recognition of Chi on exclusively single-stranded substrates has never been tested before, even though Chi is known to be recognized as the single stranded sequence 5' GCTGGTGG 3'. This study tests the effect of ssDNA oligonucleotides, having a Chi sequence (Chi+) or a single base mutant of the Chi sequence (Chio), on the enzymatic activities of RecBCD. The results obtained show that Chi is specifically recognized by RecBCD even when part of a single-stranded substrate. However its location within the single strand is important; specific recognition and distinction from the mutant sequence occurs only when Chi is in the middle of the construct, flanked by DNA at either end. The activities affected included the helicase, and consequently the exonuclease and Chi-recognition activity itself. The effects observed were also found to be dependent on the length of the oligonucleotides. The longer oligonucleotides affect the enzymatic function of RecBCD better than the shorter ones. Preincubation of DNA substrate with RecBCD abolishes the inhibitory effect of the oligonucleotides on RecBCD function however, preincubation of the oligonucleotides themselves with RecBCD does not enhance inhibition by them. This study also presents some preliminary results from photocrosslinking of oligonucleotides to RecBCD, which suggest that RecC may play an important role in binding and recognizing Chi. The results lead to the proposition of a model that explains the location-specific (flank-dependent) recognition of the Chi sequence by RecBCD and discusses the possibility of the existence of a specific site on RecBCD for Chi.