Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, the most common neoplasm in untreated HIV-1-infected individuals, and several B cell disorders. KSHV infection goes through lytic and latent phases, and the switch from latency to lytic replication is governed by viral replication and transcription activator (RTA). RTA consists of 691 amino acids, containing an N-terminal DNA-binding and a C-terminal activation domain. In the present study, polyclonal antibody against RTA was generated and evaluated. The C-terminal region of RTA (E482 similar to D691) was expressed in Escherichia coli, purified by affinity chromatography, and utilized to raise polyclonal antibody in BALB/c mice. High-affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:13,500 for ELISA and 1: 20,000 for Western blot analysis. The antibody can specifically recognize full-length RTA expressed in both E. coli and mammalian cells. Furthermore, endogenous RTA can be detected with the antibody in TPA-induced BCBL-1 cells under various conditions. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of RTA.