The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185-kDa ErbB2 receptor is rapidly (2-6 hours) induced by the HSP90 chaperone inhibitor geldanamycin (GA), whereas its downregulation and lysosomal degradation are more slowly (24 hours) induced by the proteasome inhibitor bortezomib/PS341. In PS341-treated SK-BR-3 cells, overexpressed ErbB2 coprecipitates with the E3 ubiquitin ligase c-Cbl and also with the deubiquitinating enzyme USP9x; moreover, siRNA downregulation of USP9x enhances PS341-induced ErbB2 downregulation. Because polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM)/mass spectrometry (MS) and polyubiquitin linkage-specific antibodies were used to quantitatively track K48-linked and K63-linked ErbB2 polyubiquitination following either GA or PS341 treatment of SK-BR-3 cells. MRM/MS revealed that unlike the rapid, modest (4-fold to 8-fold), and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20-fold to 40-fold) sequential increase in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces colocalization of K48-linked and K63-linked polyubiquitin with perinuclear lysosome-sequestered ErbB2. Thus, ErbB2 surface overexpression and recycling seem to depend on its polyubiquitination and deubiquitination; as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination, and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms.