The transcription factor hepatocyte nuclear factor 1 beta (HNF1 beta) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1 beta transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1 beta and its interactions with the nuclear protein import receptor, Importin-alpha. Pull-down assays demonstrated that the DNA binding domain of HNF1 beta interacted with a spectrum of Importin-alpha isoforms and deletion constructs tagged with eGFP confirmed that the HNF1 beta(KKMRRNR235)-K-229 sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-alpha using complementary biophysical techniques and have determined the 2.4 angstrom resolution crystal structure of the HNF1 beta NLS peptide bound to Importin-alpha. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1 beta and its interaction with the nuclear import protein Importin-alpha provide the basis for the development of compounds targeting transcription factor HNF1 beta via its nuclear import pathway. (C) 2016 MRC Laboratory of Molecular Biology. Published by Elsevier Inc.