Crotonaldehyde (1) is a mutagen and carcinogen, but its reactions with DNA have been only partially characterized. In a previous study, we found that substantial amounts of 2-(2-hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol, 7), the dimer of 3-hydroxybutanal (8), were released upon enzymatic or neutral thermal hydrolysis of DNA that had been allowed to react with crotonaldehyde. We have now characterized two paraldol-deoxyguanosine adducts in this DNA: N(2)-[2-(2-hydroxypropyl)-6-methyl-1,3-dioxan-4-yl]deoxyguanosine (N(2)-paraldol-dG, 13) and N(2)-[2-(2-hydroxypropyl)-6-methyl-1, 3-dioxan-4-yl]deoxyguanylyl-(5'-3')-thymidine [N(2)-paraldol-dG-(5'-3')-thymidine, 14]. Four diastereomers of N(2)-paraldol-dG (13) were observed. Their overall structures were determined by (1)H NMR, by MS, and by reaction of paraldol with deoxyguanosine and DNA. (1)H NMR data showed that two diastereomers had all equatorial substituents in the dioxane ring, while two others had an axial 6-methyl group. Preparation of paraldol with the (R)- or (S)-configuration at the 6-position of the dioxane ring and the carbinol carbon of the 2-(2-hydroxypropyl) group allowed partial assignment of the absolute configurations of N(2)-paraldol-dG (13). Four diastereomers of N(2)-paraldol-dG-(5'-3')-thymidine (14) were observed. Their overall structure was determined by (1)H NMR, MS, and hydrolysis with snake venom or spleen phosphodiesterase. Reactions of nucleosides and nucleotides with paraldol demonstrated that adducts were formed only from deoxyguanosine and its monophosphates. Experiments with DNA that had been reacted with crotonaldehyde indicated that N(2)-paraldol-dG-containing adducts in DNA are relatively resistant to enzymatic hydrolysis. The results of this study demonstrate that the reaction of crotonaldehyde with DNA is more complex than previously recognized and that stable N(2)-paraldol-dG adducts are among those that should be considered in assessing mechanisms of crotonaldehyde mutagenicity and carcinogenicity.