Cholyl adenylate is a putative intermediate for biosynthesis of cholic acid-coenzyme A (CoA) thioester conjugates by acyl-CoA synthetase. Early studies showed the conjugated acid anhydride moiety of cholyl adenylate to be reactive, attacking proteins to form protein-cholic acid adducts. In the present study, to clarify reactions of cholyl adenylate with DNA under physiological conditions, products with nucleosides were analyzed. HPLC-MS analyses indicated cholyl adenylate to primarily attack hydroxy groups of ribose moieties of nucleosides. Moreover, as speculated from UV and MS studies, exocyclic amino groups of 2′-deoxycytidine and 2′-deoxyadenosine were found to serve as targets of cholyl adenylate; the corresponding cholic amides, N 4-cholyl-2′-deoxycytidine and N6-cholyl-2′- deoxyadenosine, were formed at yields of 0.32 and 0.06%, respectively. Structures of these base modified adducts were confirmed by direct comparison with synthetic compounds obtained from coupling reactions of cholic acid with each nucleoside in the presence of dicyclohexylcarbodiimide in pyridine at 70 °C. N4-Cholyl-2′-deoxycytidine was also obtained at a level of 1.6 adducts per 105 nucleosides from enzymatic hydrolysates of calf thymus DNA reacted with cholyl adenylate. These results suggest that cholyl adenylate, released from CoA synthetase, may have some possibility as a DNA modifier in vivo.