TR3, also known as NGFI-B or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. We previously reported that TR3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. Here, we present evidence that TR3 was also induced by epidermal growth factor (EGF) and serum and was required for their mitogenic effect in lung cancer cells. Ectopic expression of TR3 in both H460 and Calu-6 lung cancer cell lines promoted their cell cycle progression and BrdU incorporation, while inhibition of TR3 expression by the small interfering RNA approach suppressed the mitogenic effect of EGF and serum. Analysis of TR3 mutants showed that both TR3 DNA binding and transactivation were required for its mitogenic effect. In contrast, they were dispensable for its apoptotic activity. Furthermore, confocal microscopy analysis demonstrated that TR3 functioned in the nucleus to induce cell proliferation, whereas it acted on mitochondria to induce apoptosis. In examining the signaling that regulates the mitogenic function of TR3, we observed that coexpression of constitutive-active MEKK1 inhibited TR3 transcriptional activity and TR3-induced proliferation. The inhibitory effect of MEKK1 was mediated through activation of Jun N-terminal kinase, which efficiently phosphorylated TR3, resulting in loss of its DNA binding. Together, our results demonstrate that TR3 is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization.TR3 (also known as NGFI-B and nur77) (6,17,44), an immediate-early response gene, is an orphan member of the steroid/thyroid/retinoid receptor superfamily, whose members mainly act as transcriptional factors to positively or negatively regulate gene expression (22,41,67). The role of TR3 in cell proliferation was suggested by numerous observations showing that its expression is rapidly induced by several mitogenic inducers, including serum growth factor, epidermal growth factor (EGF), and fibroblast growth factor (6,9,13,17,35,44,58). However, whether TR3 expression has a causal role in promoting cell proliferation remains to be illustrated. Recent evidence also indicates that the expression of TR3 is required for apoptosis. TR3 was rapidly induced by T-cell receptor signaling in immature thymocytes and T-cell hybridomas (39, 60). Overexpression of a dominant-negative TR3 protein (60) or inhibition of TR3 expression by antisense TR3 mRNA (39) inhibited T-cell-receptor-induced apoptosis, whereas constitutive expression of TR3 resulted in massive cell death (57, 64).