Transcription of lymphokine genes in activated T cells is inhibited by the immunosuppressive agents cyclosporin A and FK506, which act by blocking the phosphatase activity of calcineurin. NFAT, a DNA-binding protein required for interleukin-2 gene transcription, is a potential target for calcineurin, cyclosporin A and FK506. NFAT contains a subunit (NFATp) which is present in unstimulated T cells and which forms a complex with Fos and Jun proteins in the nucleus of activated T cells. Here we report that NFATp is a DNA-binding phosphoprotein of relative molecular mass approximately 120,000 and is a substrate for calcineurin in vitro. Purified NFATp forms DNA-protein complexes with recombinant Jun homodimers or Jun-Fos heterodimers; the DNA-binding domains of Fos and Jun are essential for the formation of the NFATp-Fos-Jun-DNA complex. The interaction between the lymphoid-specific factor NFATp and the ubiquitous transcription factors Fos and Jun provides a novel mechanism for combinatorial regulation of interleukin-2 gene transcription, which integrates the calcium-dependent and the protein-kinase C-dependent pathways of T-cell activation.