The RecBCD enzyme of Escherichia coli functions in the seemingly disparate roles of homologous recombination and the degradation of DNA. Which of these two roles it assumes is regulated by the 8-base recombination hotspot, Chi. Using double-stranded DNA substrates that are heteroduplex at the Chi locus we have established the determinants for Chi recognition. Our results show that an actively translocating RecBCD enzyme requires only the sequence information in the 5 -GCTGGTGG-3 -containing strand to recognize and to be regulated by Chi. Furthermore, the RecBCD enzyme can translocate through DNA heteroduplex bubbles as large as 22 bases, and still recognize a Chi sequence embedded in this region. This implies that recognition of Chi occurs following the unwinding of the DNA.