Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) infection is associated with increased levels of viral replication and burden in the peripheral blood and lymphoid organs. T cell activation and ensuing cellular gene activation can be critical for HIV-1 replication. The hypothesis that the nuclear factor of activated T cells (NF-AT) may influence HIV-1 replication is therefore compelling given the tight correlation of HIV-1 transcriptional induction to T cell activation. We report that certain NF-AT(Rel) family members productively bind the κB regulatory elements, synergize with NF-κB and Tat in transcriptional activation of HIV-1, and enhance HIV-1 replication in T cells. These results link regulatory factors critical to T cell commitment directly to HIV-1 replication.