BcgI is a multi-subunit restriction-modification (R-M) complex. BcgI prefers pBR322 DNA over pUC19 in a DNA cleavage reaction. Linearized pBR322 contains two BcgI recognition sites and pUC19 has only one site. To test whether two target sites are required for BcgI cleavage, one of the two sites in pBR322 was deleted, and as a result pBR322-1 became a poor substrate for BcgI. Conversely, adding a BcgI site to pUC19 makes it a much better substrate for BcgI cleavage. In addition, the BcgI (R-M) complex forms a heterohexamer in solution that is capable of interacting with two recognition sites. Our results suggest that BcgI requires two recognition sites for cleavage.