By analyzing the early genetic response of human T cells following mitogenic activation we have identified NOT, a member of the steroid/thyroid hormone family of receptors. NOT has all structural features of steroid/thyroid hormone receptors (C2C2 zinc-finger domain, ligand binding domain), but is rapidly and only very transiently expressed after cell activation, which is clearly at variance with classical steroid receptors such as glucocorticoid or estrogen receptors. NOT gene induction is independent of de novo protein synthesis, defining NOT as an immediate-early response gene. Short-lived NOT mRNA (4.2 kilobases) expression could be observed in vitro in a greater number of tissue types following activation by a variety of distinct stimuli. In vivo, NOT mRNA expression was detected exclusively in the brain, where a very strong signal was observed. By immunoblot analysis of human T cell lysates with NOT specific antisera two activation-dependent protein bands (66 and 59 kilodaltons) could be detected. NOT gene was localized to human chromosome 2q22-q23. Sequence comparison revealed that NOT is the human homolog of the murine NURR1 and rat RNR-1. Moreover NOT is closely related to NAK1/TR3, a previously identified human orphan steroid receptor. Several lines of evidence indicate that NOT and NAK1/TR3 form a distinct and exclusive subgroup of orphan steroid receptors, whose expression characteristics in vitro and in vivo resemble the expression of nonsteroid immediate-early transcription factors such as jun and fos. NOT and NAK1/TR3 thus may function as general coactivators of gene transcription rather than participate in the induction of specific target genes, as is the case with classical steroid receptors.