The structure and conformation of guanosine 5’-monophosphate in hydrogen-bonded quartet assemblies have been investigated by use of the vanadyl ion (VO2+) as a paramagnetic probe for EPR and electron nuclear double resonance (ENDOR) spectroscopy. EPR spectrometric titrations showed that the stoichiometry of VO2+:nucleotide binding was 1:2. Proton ENDOR absorptions of the vanadyl-nucleotide complex indicated the presence of only axially coordinated H2O in the inner coordination sphere. This result and the observation of a P-31 superhyperfine coupling pattern of 1:2:1 relative intensity in EPR spectra established that chelation of VO2+ occurs through phosphate groups in a bidentate fashion with oxygen atoms positioned in the equatorial plane to form a [VO(GMP)(2)(H2O)] complex. Specific assignments of proton ENDOR absorptions were made for the purine H(8) and a ribose OH on the basis of deuterium substitution. Conditions under which [VO(GMP)(2)(H2O)] was incorporated predominantly into quartets or stacked quartets at 1 degrees C were established by NMR. Proton ENDOR spectra of frozen solutions showed identical splittings for [VO(GMP)(2)(H2O)] as a monomer complex or when incorporated predominantly into quarters or stacked quartets. From the principal hyperfine coupling components, dipolar electron-proton distances were calculated and applied as constraints in computer-based torsion angle search calculations. The results showed that the guanine base acquires an anti conformation while the ribose moiety could be accommodated only by a C3’-endo conformation. The results are discussed with reference to recent X-ray crystallographic and NMR studies of G-quartets formed by synthetic oligonucleotide strands of DNA and RNA.