We have previously reported that the hepatitis B virus X protein (HBx) activates nuclear factor of activated T cells (NF-AT), a key regulator of the immune system, by a calcium/calcineurin-dependent pathway, involving dephosphorylation and nuclear translocation of this transcription factor. In addition, we showed that HBx synergizes with potent calcium-mobilizing stimuli to activate NF-AT-dependent transcription, suggesting that additional mechanisms might also be operative in the activation of NF-AT by HBx. Here we demonstrate that HBx activates the NH(2)-terminal transcription activation domain (TAD) of NF-AT1 by a mechanism involving protein-protein interaction. Targeting of HBx to the nucleus did not affect its ability to induce the transcriptional activity of NF-AT1. In contrast, mutations of HBx affecting its functional interaction with general transcription factors abrogated the HBx-induced activity of NF-AT1. Together these results indicate that HBx may exert its function by acting as a nuclear coactivator of NF-AT1.