Oligodeoxynucleotides (ODNs) containing 5-formyl-2'-deoxycytidine (fC) were synthesized by the phosphoramidite method and subsequent oxidation with sodium periodate. The stabilities of duplexes containing A, G, C or T opposite fC were studied by thermal denaturation. It was found that fC:A, fC:C or fC:T base pairs significantly reduce the thermal stabilities of duplexes. Next, single nucleotide insertion reactions were performed using ODNs containing fC as templates and the Klenow fragment of Escherichia coli DNA polymerase I. It was found that: (i) insertion of dGMP opposite fC appears to be less efficient relative to insertion opposite 5-methyl-2'-deoxycytidine (mC); (ii) dAMP is misincorporated more frequently opposite fC than mC, although the frequency of misincorporation seems to be dependent on the sequence; (iii) TMP is misincorporated more frequently opposite fC than mC. These results suggest that fC may induce the transition mutation C.G-->T.A and the transversion mutation C.G-->A.T during DNA synthesis.