The average conformations of guanylyl(3′-5′)cytidine (GpC) and cytidylyl(3′-5′)guanosine (CpG) in neutral aqueous solution have been investigated by proton-proton and proton-phosphorus coupling constants and by T1 relaxation time measurements at 250 MHz. The temperature effect on the chemical shifts and coupling constants has been studied in the temperature interval between 25°C and 65°C. Apart from the H-5 of the pC residue in GpC, which shows a downfield shift of about 0.18 ppm, no dramatic changes in chemical shift have been observed for other protons. On the other hand, the couplings JH-H and JP-H of both dinucleoside monophosphates are quite sensitive to temperature. The results indicate that the temperature effect is larger for GpC than for CpG. The N conformer is favoured at low temperature. At 25°C its proportion is preponderant and is more important for the C than for the G residue in both dimers. When the temperature increases from 25°C to 65°C, the N proportions decrease and approach those of the corresponding mononucleotides. The largest change in N/S equilibrium with temperature is observed in the Gp residue of GpC. The conformers gauche-gauche and gauche– (or gauche+) are favoured in both dinucleoside monophosphates. At 25°C, the proportion of the gg rotamer of pC in GpC is almost equal to 100%. This indicates that the rotation about the phosphodiester linkage of this molecule is very reduced. The rotamer distribution of the 3′ residue exocyclic groups of both GpC and CpG is virtually unchanged, while that of the 5′ residues is notably affected by the temperature variation. The conformation of the base around the glycosidic bond was studied by T1 proton relaxation time measurement before and after deuteration of H-8 of the G residue. In the case of GpC, the syn conformation is predominant in the Gp, while the anti conformation is highly favoured in the pC residue. In the case of CpG, the guanine base is close to the sugar of the Cp moiety at least part of the time. The syn and anti conformations appear equally probable in both residues. As far as the temperature effect and conformational analysis are concerned, both residues of CpG behave similarly to the corresponding mononucleotides, suggesting a great flexibility of this dimer.