An algorithm for designing a combinatorial library comprehensively representing all hexamer palindrome sequences at uniquely defined sites is described. The expected size for such a library of 64 possible hexamer palindromes is 384 bases, which is reduced to 266 bases spread over 8 oligonucleotides through a linear overlap of rationally selected hexamer palindromes. The single stranded oligonucleotides of the designed sets were chemically synthesized and converted into corresponding duplex dimers using PCR primer-dimer method. The utility of these duplex oligomers for identifying cleavage sites of restriction enzymes recognizing hexamer palindromes has been demonstrated using some representative enzymes. The library is also useful for screening restriction enzymes with tetramer cleavage sites and identifying the "star" sites of restriction enzymes. The sets of oligonucleotides with high information content, though designed for direct and unambiguous characterization of cleavage sites of isolated restriction enzymes, have potential applications as templates for characterizing sequence selective binding and interaction of small molecules nucleic acid.