The commercially available Jak2 inhibitor, α-cyano-3,4-dihydroxy-N- benzylcinnamide (AG490), has been used extensively to study Jak2 kinase function. While α-cyano-3,4-dihydroxy-N-benzylcinnamide is a potent Jak2 inhibitor, it can inhibit a number of other kinase signaling pathways as well. To circumvent this problem, we sought to identify novel small molecule inhibitors of Jak2 tyrosine kinase activity. For this, we constructed a homology model of the Jak2 kinase domain and identified solvent accessible pockets on the surface of the structure. Using the DOCK program, we tested 6451 compounds of known chemical structure in silico for their ability to interact with a pocket positioned adjacent to the activation loop. We attained the top seven scoring compounds from the National Cancer Institute and tested their ability to inhibit Jak2 autophosphorylation in vitro. Using Western blot analysis, we found that one of the compounds, 1,2,3,4,5,6-hexabromocyclohexane, was able to potently, and directly, inhibit Jak2 autophosphorylation. Characterization of this compound revealed that it inhibits Jak2 tyrosine autophosphorylation in both a time- and concentration-dependent manner. It greatly reduced growth hormone-mediated Jak2 autophosphorylation but did not block autophosphorylation of the epidermal growth factor receptor. Furthermore, doses as high as 100 μM were not toxic to cells as measured by their ability to exclude propidium iodide. As such, we believe that this compound could serve as a lead compound for a new generation of Jak2 inhibitors and, perhaps, be useful in elucidating the mechanisms of Jak2 kinase function. © 2005 American Chemical Society.