Sequence and structural anomalies in the gene for androgen receptor (AR) or its protein are associated with a range of clinical manifestations. Observations from living cells have shown that AR translocates to the nucleus upon ligand binding, where it forms typical ‘nuclear foci’ that are considered as sites of gene transcription. Recently, we reported the ligand-mediated association of AR with mitotic chromatin and suggested its role in ‘transcription memory’, proposing a ‘biopit model’. In the present study, we show that each of the AR domains is obligatory for its association with mitotic chromatin and also that full-length AR is necessary for efficient association. In addition, deletion or point mutations in bipartite nuclear localization signal (NLS) revealed impaired localization, ‘nuclear foci’ formation and abolished AR binding with mitotic chromatin. Interestingly, well-characterized AR-NLS mutants associated with the manifestation of pathological conditions (prostate cancer and androgen-insensitivity syndrome) exhibited differential behaviour on mitotic chromatin and also impaired receptor localization and ‘nuclear foci’ formation. Finally, we report that, in addition to its functions in nuclear import, DNA binding, acetylation, N/C-termini interactions and transactivation, the AR-NLS region also functions as ‘mitotic chromatin binding-determining region’ and has a novel role in the regulation of the AR association with mitotic chromatin.