The enthalpy of hydrogen-bond formation between guanine (G) and cytusine (C) in o-dichlorobenzene and in chloroform at 25Β°C has been determined by direct calorimetric measurement. We derivatized 2'-deoxyguanosine and 2'-deoxycytidine at the 5'-and 3'-hydroxyls with triisopropylsilyl groups; these group increase the solubility of the nucleic acid bases in nonaqueous solvents. Such derivatization also prevents the r i b hydroxyls from forming hydrogen bonds. Consequently, hydrogen-bond formation in our system is primarily between the bases, and to a lesser extent, between base and solvent, and can be measured directly with calorimetry. To obtain the data on baae-pair formation, we first took into account the contributions from self-association of each base, and where possible, have determined the AH of self-association. From isoperibolic titration dorimetry, our measured AH of C2 formation in chloroform is -1.7 kcal/mol of C. Our measured AH of C : G base-pair formation in o-dichlorobenzene is -6.65 0.32 kcal/mol. Since o-dichlorobenzene does not form hydrogen bonds, the AH of C : G base-pair formation in this solvent represents the AH of the hydrogen-bonding interaction of C with G in a nonassociating solvent. In contrast, our measured AH of C : G basepair formation in chloroform is -5.77 f 0.20 kd/mol; thus, the absolute value of the enthalpy of hydrogen bonding in the C : G base pair is greater in o-dichlorobenzene than in chloroform. Since chloroform is a solvent known to form hydrogen bonds, the decrease in enthalpic contribution to C : G base pairing in chloroform is due to the formation of hydrogen bonds between the bases and the solvent. The AH of hydrogen bonding of G with C reported here differs from previous indirect estimates: Our measurements indicate the AH is 50% less in magnitude than the AH based on spectroscopic mensurements of the extent of interaction. We have also observed that the enthalpy of hydrogen bonding of C with G in chloroform is greater when G is in excess than when C is in excess. This increased heat is due to the formation of C : G, > complexes that we have observed using H-nmr. Although C:G2 structures have previously been observed in triplestranded polymeric nucleic acids, higher order structures have not been observed between C and G monomers in nonaqueous solvents until now. By using monomers as a model system to investigate hydrogen-bonding interactions in DNA and RNA, we have obtained the following results: A direct measurement of the AH of hydrogen bonding in the C : G complex in two nonaqueous solvents, and the first observation of C : G,, , complexes between monomers. These results reinforce the importance of hydrogen bonding in the stabilization of various nucleic acid secondary and tertiary structures.