Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is a large transcriptional regulator essential for EBV-mediated immortalization of B lymphocytes. We previously identified interactions between EBNA-3C and two cellular transcription factors, J kappa and Spi proteins, through which EBNA-3C regulates transcription. To better understand the contribution of these interactions to EBNA-3C function and EBV latency, we examined whether they are conserved in the homologous proteins of nonhuman primate lymphocryptoviruses (LCVs), which bear a strong genetic and biological similarity to EBV. The homologue of EBNA-3C encoded by the LCV that infects baboons (BaLCV) was found to be only 35% identical in sequence to its EBV counterpart. Of particular significance, this homology localized predominantly to the N-terminal half of the molecule, which encompasses the domains in EBNA-3C that interact with J kappa and Spi proteins. Like EBNA-3C, both BaLCV and rhesus macaque LCV (RhLCV) 3C proteins bound to J kappa and repressed transcription mediated by EBNA-2 through its interaction with J kappa. Both nonhuman primate 3C proteins were also able to activate transcription mediated by the Spi proteins in the presence of EBNA-2. Like EBNA-3C, a domain encompassing the putative basic leucine zipper motif of the BaLCV-3C protein directly interacted with both Spi-1 and Spi-B. Surprisingly, a recently identified motif in EBNA-3C that mediates repression was not identifiable in the BaLCV-3C protein. Finally, although the C terminus of BaLCV-3C bears minimal homology to EBNA-3C, it nonetheless contains a C-terminal domain rich in glutamine and proline that was able to function as a potent transcriptional activation domain, as does the C terminus of EBNA-3C. The conservation of these functional motifs despite poor overall homology among the LCV 3C proteins strongly suggests that the interactions of EBNA-3C with J kappa and Spi do indeed play significant roles in the life cycle of EBV.