Uridylyl-(3'→5')-uridine (UpU) was synthesized via two different routes, i.e. by condensation of 2', 5'-di-O-trityluridine (I) with 2', 3'-di-O-acetyluridine 5'-phosphate (IV) (Route A) and by condensation of 2', 3'-di-O-acetyluridine (VI) with 2', 5'-di-O-trityluridine 3'-phosphate (II) (Route B). The acid treatments to remove trityl groups were performed with 80% acetic acid at both 37° and 100° in several time intervals. The yields of the final product, UpU, and the amounts of uridylyl-(2'→5')-uridine contained in the UpU were compared.