NASP (nuclear autoantigenic sperm protein) is a linker histone-binding protein found in all dividing cells that is regulated by the cell cycle (Richardson, R. T., Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield, M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386), and in the nucleus linker histones not bound to DNA are bound to NASP (Alekseev, O. M., Bencic, D. C., Richardson R. T., Widgren E. E., and O'Rand, M. G. (2003) J. Biol. Chem. 278, 8846-8852). In mouse spermatogenic cells tNASP binds the testis-specific linker histone H1t. Utilizing a cross-linker, 3,3'-dithiobissulfosuccinimidyl propionate, and mass spectrometry, we have identified HSP90 as a testis/embryo form of NASP (tNASP)-binding partner. In vitro assays demonstrate that the association of tNASP with HSP90 stimulated the ATPase activity of HSP90 and increased the binding of H1t to tNASP. HSP90 and tNASP are present in both nuclear and cytoplasmic fractions of mouse spermatogenic cells; however, HSP90 bound to NASP only in the cytoplasm. In vitro nuclear import assays on permeabilized HeLa cells demonstrate that tNASP, in the absence of any other cytoplasmic factors, transports linker histones into the nucleus in an energy and nuclear localization signal-dependent manner. Consequently we hypothesize that in the cytoplasm linker histones are bound to a complex containing NASP and HSP90 whose ATPase activity is stimulated by binding NASP. NASP-H1 is subsequently released from the complex and translocates to the nucleus where the H1 is released for binding to the DNA.