We report the first observation of a spin-labeled ds 23-mer oligonucleotide by high-field electron spin resonance (ESR) and demonstrate that it interacts with AP endonuclease, the key enzyme in DNA abasic site repair. The spin labeled 23-mer with a U at position 12 of the upper strand is processed by uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, A-P endo binds and cleaves when the label is 3' but not 5' to the abasic site. These results confirm. that the disposition of the bases immediately upstream of the abasic site is particularly critical for cleavage by A-P endo, and establish that DNA-protein interactions in this important enzyme can be examined using spin-labeled substrates.