The interleukin-1 receptor-associated kinase 1 (IRAK-1) is an important adapter in the signaling complex of the Toll/interleukin-1 (IL-1) receptor family. Formation of the signaling IL-1 receptor complex results in the activation and hyperphosphorylation of IRAK-1, which leads to a pronounced shift of its apparent molecular mass in gel electrophoresis. Presently, the individual residues phosphorylated in IRAK-1 and the consequences for IRAK-1 function are unknown. We define sequential phosphorylation steps in IRAK-1, which are, in vitro, autophosphorylation. First, IRAK-1 is phosphorylated at Thr