In this work, we examined the DNA sequence preference of gamma-radiation-induced DNA damage in purified DNA sequences after heat treatment. DNA was fluorescently end-labeled and gamma-radiation-induced DNA cleavage was examined using capillary electrophoresis with laser-induced fluorescence detection. Our findings provide evidence that gamma-radiation-induced DNA damage to end-labeled DNA is nonrandom and has a sequence preference. The degree of cleavage was quantified at each nucleotide, and we observed that preferential cleavage occurred at C nucleotides with lesser cleavage at G nucleotides, while being very low at T nucleotides. The differences in percentage cleavage at individual nucleotides ranged up to sixfold. The DNA sequences surrounding the most intense radiation-induced DNA cleavage sites were examined and a consensus sequence 5′-AGGC*C (where C* is the cleavage site) was found. The highest intensity gamma-radiation-induced DNA cleavage sites were found at the dinucleotides, 5′-GG*, 5′-GC*, 5′-C*C and 5′-G*G and at the trinucleotides, 5′-GG*C, 5′-TC*A, 5′-GG*G and 5′-GC*C. These findings have implications for our understanding of ionizing radiation-induced DNA damage.