NF-κB-inducing kinase (NIK) is required for NF-κB activation based on the processing of NF-κB2 p100. Here wereport a novel mechanism of NIK regulation involving the chaperone 90 kDa heat shock protein (Hsp90) and autophagy.Functional inhibition of Hsp90 by the anti-tumor agent geldanamycin (GA) efficiently disrupts its interaction with NIK,resulting in NIK degradation and subsequent blockage of p100 processing. Surprisingly, GA-induced NIK degradationis mediated by autophagy, but largely independent of the ubiquitin-proteasome system. Hsp90 seems to be specificallyinvolved in the folding/stabilization of NIK protein, because GA inhibition does not affect NIK mRNA transcription andtranslation. Furthermore, Hsp90 is not required for NIK-mediated recruitment of the a subunit of IκB kinase to p100, akey step in induction of p100 processing. These findings define an alternative mechanism for Hsp90 client degradationand identify a novel function of autophagy in NF-κB regulation. These findings also suggest a new therapeutic strategyfor diseases associated with p100 processing. NF-κB activation inducing kinase (NIK) is required for NF-κB activation based on the processing of NF-κB2 p100. Here wereport a novel mechanism of NIK regulation involving the chaperone 90 kDa heat shock protein (Hsp90) and autophagy. Functional inhibition Surprisingly, GA-induced NIK degradation mediated by autophagy, but largely independent of the ubiquitin-proteasome system . Hsp90 seems to be specifically involved in folding / stabilization of NIK protein, because GA inhibition does not affect NIK mRNA transcription and translation. Furthermore, Hsp90 is not required for NIK-mediated recruitment of the subunit of IκB kinase to p100, akey step in induction of p100 processing. These findings define an alternative mechanism for Hsp90 client degradation and identify a novel function of autophagy in NF-κB regulation. These f indings also suggest a new therapeutic strategy for diseases associated with p100 processing.