Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) was studied as both a 2'-deoxyribosyl 5'-triphosphate derivative and as a template incorporated into an oligonucleotide.Using a novel Pyrosequencing assay zebularine acted as cytosine analog and was incorporated into DNA with a template pairing profile most similar to cytosine, pairing with greatest efficiency opposite guanine in the template strand. Guanine was incorporated with greater affinity than adenine opposite a zebularine in the template strand. Computer modeling of basepairing structures supported a better fit of zebularine opposite guanine than adenine. Zebularine