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Base damage in alpha DNA from irradiated monkey CV-1 cells was determined by measuring release of 5'-32P-end labelled DNA fragments after digestion with endonuclease III of E. coli. The frequency and base sequence locations of the enzyme-sensitive sites were determined. Fragments were released from irradiated DNA at sequence sites of pyrimidines and guanines. The time for repair of half the single strand breaks was approximately 1.5 h. Repair of base damage as judged from loss of enzyme-sensitive sites in DNA was slower, with more than half of the damaged bases still detectable after 4 h of repair. Two important changes in the pattern of fragment release from DNA were produced when small radiation doses preceded the large ones needed to produce measurable DNA strand breaks and base damage. 5 Gy to cells incubated several hours before 320 Gy increased by five-fold the abundance of small DNA fragments with 3'-phosphoryl termini detected in high-resolution denaturing gels. These increases were detectable with doses as small as 0.2 Gy and were accompanied by the appearance of new species of DNA fragments of intermediate mobility at specific locations in the base sequence. The patterns resemble those produced by digesting DNA from heavily irradiated cells with endonuclease III.