To study the binding of pranlukast to hRKIP and its regulatory role in the Raf1/MEK/ERK signal pathway. NMR and fluorescence experiments demonstrated hRKIP could bind pranlukast with a binding constant of 1016 mM(-1). Residues (Y81, S109 and Y181) on the conserved ligand-binding pocket of hRKIP played a crucial role in binding pranlukast, and their mutations reduced the binding affinity more than 85 %. Furthermore, 25 mu M pranlukast could up-regulate the ERK phosphorylation by about 17 %. Pranlukast may be used as a potential drug precursor for treating hRKIP involved diseases.