Human immunodeficiency virus type 1 (HIV-1) Tat affects cellular gene expression through modulation of the activity of different transcription factors. Here, the role of Tat in the cooperation between nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) transcription factors was investigated. Constitutive or transient Tat expression in Jurkat T cells enhanced cooperative NFAT/AP-1- but not AP-1-dependent transcription independent of its ability to transactivate the HIV-1 LTR. The enhancing effect of Tat took place after nuclear translocation of NFAT. Furthermore, transactivation of an NFAT/AP-1 reporter by transfection of NFAT and c-Jun was strongly enhanced by simultaneous Tat transfection. Moreover, intracellular Tat expression increased the binding of NFAT/AP-1 complexes to the interleukin 2 promoter without significantly altering NFAT- and AP-1-independent binding. HIV-1 Tat interacted with NFAT but not c-Jun. These results indicate that Tat interacts with NFAT, affecting its cooperation with AP-1, without altering independent binding of these transcription factors to DNA.