The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3D(pol). Using in vitro assay systems previously described for poliovirus RNA polymerase 3D(pol) (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3D(pol) is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn(2+), and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)(15) primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5\' end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.