The influence of structural changes of an abasic site in duplex DNA on noncovalent and site-directed spin labeling (NC-SDSL) of the spin label ç were examined with electron paramagnetic resonance (EPR) spectroscopy. The binding affinities of ç to sixteen different DNA duplexes containing all possible sequences immediately flanking the abasic site were determined and the results showed that the binding of ç is highly flanking-sequence dependent. In general, a 5'-dG nucleotide favors the binding of the spin label. In particular, 5'-d(G__T) was the best binding sequence whereas 5'-d(C__T) showed the lowest affinity. Changing the structure of the abasic site linker from a tetrahydrofuran analog (F) to the anucleosidic C(3)-spacer (C(3)) does not appreciably affect the binding of ç to the abasic site. For efficient binding of ç, the abasic site needs to be located at least four base pairs away from the duplex end. Introducing a methyl substituent at N3 of ç did not change the binding affinity, but a decreased binding was observed for both N3-ethyl and -propyl groups. These results will guide the design of abasic site receptors and spin label ligands for NC-SDSL of nucleic acids.