2'-Deoxy-lin-benzoadenosine has been synthesized via reductive deoxygenation of 2-(beta-D-ribofuranosyl)-8-(methylthio)imidazo[4,5-g]quinazoline. The 5'-mono-, 5'-di-, and 5'-triphosphates have been prepared by chemical and/or enzymatic methods. The 5'-diphosphate was found to be a substrate for phosphorylation by pyruvate kinase and was compared with various natural and extended substrates in kinetic assays. When 2'-deoxy-lin-benzoadenosine 5'-triphosphate was tested in a nick-translation experiment with Escherichia coli DNA polymerase I, a very low level of 32P incorporation from [alpha-32P]TTP into poly[d(AT)] was observed. Nearest-neighbor analysis indicated that the analogue was not significantly incorporated into internal positions in the polymer. In DNA-sequencing reactions, the analogue caused chain termination at adensine residues, although termination was less uniform and less efficient than that with 2',3'-dideoxy-ATP. These experiments show that lin-benzoadenine can form a widened Watson-Crick base pair with thymine. They strongly suggest, though they do not prove, that the enzyme is able to attach the analogue to DNA.