Very highly degenerate primers with short specific 3' anchor sequences and 5' adaptors were used in conjunction with nested specific primers to amplify large numbers of unknown insertion junctions of the insect retrotransposon Woot, using genomic DNA as template for the polymerase chain reaction (PCR). This technique, sometimes referred to as universal PCR, is a powerful method for molecular characterization of transposon insertions into genomes, and more generally for short-distance chromosome walking through unknown DNA. Twenty-four unique insertion junctions were cloned and sequenced from two strains of Tribolium castaneum and one strain of T. freemani. Inspection of these sequences revealed that integration of the Woot retrotransposon is cued by the insertion target motif, GTAC, in both species.