The 5'-triphosphates of 2'-hydroxymethyluridine (2'-homouridine) and 2'-hydroxyethyluridine were prepared from the corresponding acetyl-protected nucleosides by initial phosphitylation with 2-chloro-5,6-benzo-1,2,3-dioxaphosphorin-4-one. 2'-Acetamidouridine 5'-triphosphate was prepared in an analogous fashion from uridine 2'-C-, 3'-O-gamma-butyrolactone, in which the 3'-hydroxyl group is internally protected as the lactone. Subsequent treatment with ammonia gave the required acetamido triphosphate. All three triphosphates were investigated as substrates for T7 RNA polymerase and a Y639F mutant of this enzyme. 2'-Homouridine triphosphate was found to be a substrate for the wild-type enzyme in the presence of manganese and was specifically incorporated into short RNA transcripts (20 and 21 nucleotides in length). The presence of the analogue within the transcripts was confirmed through its resistance to alkaline hydrolysis. Gel electrophoretic analysis also showed that 2'-homouridine could be multiply incorporated into a transcript with a length of 75 nucleotides. This is the first report of a 2'-deoxy-2'-alpha-C-branched nucleoside 5'-triphosphate acting as a substrate for T7 RNA polymerase. The 2'-hydroxyethyl- and 2'-acetamido -uridine triphosphates were not substrates for the enzymes.