Porcine circovirus type 2 (PCV2) is a non-enveloped, single-stranded, circular DNA virus. In situ hybridization and PCR assays have detected PCV2 DNA in multiple organs and cell types from infected pigs; however, it is not clear if this represents replicating virus or virion DNA. We describe the development of a single-tube RT-PCR assay to differentiate PCV2 replication products and virus DNA. Primers targeted to the open-reading frame 2 (ORF2) of PCV2 were designed to amplify both virus DNA (984 bp) and the spliced Cap mRNA (594 bp). The 984 bp fragment, but not the 594 bp fragment, was amplified from PCV2 stock, confirming that the spliced Cap mRNA was not present in the PCV2 stock. The 594 bp fragment was amplified from DNase-treated RNA extracted from PCV2-infected PK-15 cells, and was detected as early as 14 h post-infection. No products were amplified from either the PCV1 stock or PCV1-infected PK-15 cells, or from cells infected with UV-inactivated PCV2. Therefore, the presence of the 594 bp fragment is specific for PCV2 replication. This assay will be useful in assessing cell populations that support PCV2 replication in vivo or in vitro and advance the understanding of PCV2 replication and pathogenesis.