Mutagenized human 293 cells containing an interleukin-1 (IL-1)-regulated herpes thymidine kinase gene, selected in IL-1 and gancyclovir, have yielded many independent clones that are unresponsive to IL-1. The four clones analyzed here carry recessive mutations and represent three complementation groups. Mutant A in complementation group I1 lacks IL-1 receptor-associated kinase (IRAK), while the mutants in the other two groups are defective in unknown components that function upstream of IRAK. Expression of exogenous IRAK in I1A cells (I1A-IRAK) restores their responsiveness to IL-1. Neither NFkappaB nor Jun kinase is activated in IL-1-treated I1A cells, but these responses are restored in I1A-IRAK cells, indicating that IRAK is required for both. To address the role of the kinase activity of IRAK in IL-1 signaling, its ATP binding site was mutated (K239A), completely abolishing kinase activity. In transfected I1A cells, IRAK-K239A was still phosphorylated upon IL-1 stimulation and, surprisingly, still complemented all the defects in the mutant cells. Therefore, IRAK must be phosphorylated by a different kinase, and phospho-IRAK must play a role in IL-1-mediated signaling that does not require its kinase activity.