New immunosuppressive compounds with less systemic toxicity that could replace calcineurin inhibitors are urgently needed. For identification of specific inhibitors of JAK3, a potential new drug target, from large chemical libraries we developed a cell-based screening system. TEL JAK fusion proteins composed of an oligomerization domain of TEL and kinase and/or pseudokinase domains of JAKs provided constitutive activation of JAKs without receiving a signal from the cytokine receptors. These fusion proteins also induced STAT5b phosphorylation in the absence of cytokine receptors. Both the kinase and pseudokinase domains of JAKs were required for full activation of the JAKs, and four copies of STAT5 response elements provided the greatest luciferase activity. The sensitivity and specificity of the system was evaluated using specific JAK3, JAK2, or MEK inhibitors. Thus, we generated a receptor-independent, cell-based selective screening system for specific JAK3 inhibitors, which is easily convertible to a high-throughput screening platform. (C) 2010 Elsevier B.V. All rights reserved.