This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique. In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5'-end, followed by ligation of a one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky end as the digested genomic DNA fragments, except that the 5'-overhang base overlaps the corresponding 3'-end base of the restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3'-terminal base of the genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor in which the 5'-terminal base was not consistent with the corresponding 3'-terminal base. Using this technique, the 3'- and 5'-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined.