NF-kappaB-inducing kinase (NIK) is required for NF-kappaB activation based on the processing of NF-kappaB2 p100. Here we report 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 degradation is mediated by autophagy, but largely independent of the ubiquitin-proteasome system. Hsp90 seems to be specifically involved in the 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 alpha subunit of IkappaB kinase to p100, a key 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-kappaB regulation. These findings also suggest a new therapeutic strategy for diseases associated with p100 processing.