Exposure to formaldehyde results in the formation of DNA-protein cross-links (DPCs) as a primary genotoxic effect. Although DPCs are biologically important and eight amino acids have been reported to form stable adducts with formaldehyde, the structures of these cross-links have not yet been elucidated. We have characterized formaldehyde-induced cross-links of Lys, Cys, His, and Trp with dG, dA, and dC. dT formed no cross-links, nor did Arg, Gln, Tyr, or Asn. Reaction of formaldehyde with Lys and dG gave the highest yield of cross-linked products, followed by reaction with Cys and dG. Yields from the other coupling reactions were lower by a factor of 10 or more. Detailed structural examination by NMR and mass spectrometry established that the cross-links between amino acids and single nucleosides involve a formaldehyde-derived methylene bridge. Lys yielded two additional products with dG in which the linking structure is a 1,N(2)-fused triazino ring. The Lys cross-linked products were unstable at ambient temperature. Reactions between the reactive N(alpha)-Boc-protected amino acids and the trinucleotides d(T(1)B(2)T(3)) where B(2) is the target base G, A, or C and reactions between dG, dA and dC and 8-mer peptides containing a single reactive target residue at position 5 yielded cross-linked products with structures inferred from high resolution mass spectrometry and fragmentation patterns that are consistent with those between N(alpha)-Boc-protected amino acids and single nucleotides rigorously determined by NMR studies. These structures will provide a basis for investigation of the characteristics and properties of DPCs formed in vivo and will be helpful in identifying biomarkers for the evaluation of formaldehyde exposure both at the site of contact and at distant sites.