Identification of regulatable mechanisms by which transcription factor NF-E2 p45-related factor 2 (Nrf2) is repressed will allow strategies to be designed that counter drug resistance associated with its upregulation in tumours that harbour somatic mutations in Kelch-like ECH-associated protein-1 (Keap1), a gene that encodes a joint adaptor and substrate receptor for the Cul3-Rbx1/Roc1 ubiquitin ligase. We now show that mouse Nrf2 contains two binding sites for β-transducin repeat-containing protein (β-TrCP), which acts as a substrate receptor for the Skp1-Cul1-Rbx1/Roc1 ubiquitin ligase complex. Deletion of either binding site in Nrf2 decreased β-TrCP-mediated ubiquitylation of the transcription factor. The ability of one of the two β-TrCP-binding sites to serve as a degron could be both increased and decreased by manipulation of glycogen synthase kinase-3 (GSK-3) activity. Biotinylated-peptide pull-down assays identified DSGIS(338) and DSAPGS(378) as the two β-TrCP-binding motifs in Nrf2. Significantly, our pull-down assays indicated that β-TrCP binds a phosphorylated version of DSGIS more tightly than its non-phosphorylated counterpart, whereas this was not the case for DSAPGS. These data suggest that DSGIS, but not DSAPGS, contains a functional GSK-3 phosphorylation site. Activation of GSK-3 in Keap1-null mouse embryonic fibroblasts (MEFs), or in human lung A549 cells that contain mutant Keap1, by inhibition of the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB)/Akt pathway markedly reduced endogenous Nrf2 protein and decreased to 10-50% of normal the levels of mRNA for prototypic Nrf2-regulated enzymes, including the glutamate-cysteine ligase catalytic and modifier subunits, glutathione S-transferases Alpha-1 and Mu-1, haem oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Pre-treatment of Keap1(-/-) MEFs or A549 cells with the LY294002 PI3K inhibitor or the MK-2206 PKB/Akt inhibitor increased their sensitivity to acrolein, chlorambucil and cisplatin between 1.9-fold and 3.1-fold, and this was substantially attenuated by simultaneous pre-treatment with the GSK-3 inhibitor CT99021.