Cleavage site of HphI, a type IIS restriction endonuclease, were determined by using the mutant plasmids carding various nucleotide sequences around the cleavage site. Linear, radioactively labeled plasmid DNA was reacted with HphI under the standard assay conditions, and exact cleavage sites and relative cleavage activities at the cleavage sites were determined by sizing and quantitation of the resultant cleavage products. Contrary to the previous views, our results suggest that cleavage site can be shifted depending on the nucleotide sequences between the recognition and cleavage sites, rather than restricted to 8 base pairs away from the recognition sequence. In addition, there is no A:T or T:A base pair preference at the cleavage sites. Also, it was found that the cleavage activity was asymmetrical; the numbers of cleavage sites on two strands were different. All the plasmids having -TATA- from +4 to +7 show that cleavage site is located 9 bases away from the recognition in the upper strand, while others having TATC- or -TACC- show extra cleavage activity at the 8th base pair. The characteristic cleavage pattern of each plasmid was not affected by changing environmental conditions, such as ionic strength, glycerol concentration, pH, EtBr concentration, and temperature. These cleavage site shifts and asymmetrical cleavages can be interpreted as the results form the sequence dependent conformation changes of DNA double helix.