MSL1 protein regulates global histone H4 acetylation at residue K16 in stem and cancer cells, through interaction with KAT8. The functional significance of mammalian MSL1 isoforms, involved in various protein interactions, is poorly understood. We report the identification of a novel nuclear localization signal (NLS), common to all MSL1 isoforms, in addition to previously known bipartite NLS, located in domain PEHE. Isoforms having both NLS localize to sub-nuclear foci where they can target co-chaperone protein TTC4. However, all MSL1 isoforms also have ability to affect H4K16 acetylation. Thus, presence of two NLS in MSL1 protein can mediate activity of KAT8 in vivo. J. Cell. Biochem. 115: 1967–1973, 2014. © 2014 Wiley Periodicals, Inc.