Calf thymus single-stranded DNA was modified with 2-bromoacrolein (2BA), a genotoxic metabolite of tris(2,3-dibromopropyl)phosphate. This DNA was used as a template for in vitro DNA replication by T7-polymerase and Klenow fragment of Escherichia coli DNA polymerase I. Increasing levels of 2BA modification led to decreased DNA synthesis as measured by [methyl-3H]dTTP incorporation. M13 mp19 single-stranded DNA template modified with 2BA was used to determine the sites of termination of DNA replication by T7 polymerase and Klenow fragment of Escherichia coli DNA polymerase I. It was found that DNA replication stopped frequently before and occasionally opposite deoxycytidine nucleotides. These results indicated that an as yet unidentified deoxycytidine:2BA adduct may have been formed in the reaction of 2BA with M13 DNA. To investigate if such adducts were formed, we reacted 2BA with deoxycytidine in vitro at pH 4.4, and putative deoxycytidine:2BA adducts were isolated by high-performance liquid chromatography. They were characterized by 1H and 13C nuclear magnetic resonance and with fast atom bombardment mass spectrometry as two diastereomeric 3-bromo-7-(beta-D-deoxyribofuranosyl)- 3,4-dihydro-2-hydroxy-(2H,7H)[1,6-a]pyrimidin-6-one adducts and a 3-bromo-7-(beta-deoxyribofuranosyl)-(4H,7H)-pyrimido[1,6-a]pyrimidin-6 -one adduct. Only the latter adduct, however, was formed in the reaction of 2BA with calf thymus single-stranded DNA in vitro. Tris(2,3-dibromopropyl)phosphate is clastogenic. Because clastogenicity may result from DNA adducts that block replication, the role of the presently identified deoxycytidine adducts of the reaction metabolite 2BA in the clastogenicity of tris(2,3-dibromopropyl)phosphate is discussed.