We have reported previously that the prototype foamy virus (PFV) integrase (IN) has a strong nuclear localization signal (NLS) in its C-terminal domain, in particular in a region of aa 306-334 including highly karyophilic arginines or lysines at positions 308, 313, 318, 324, and 329. In this study, we used various mutants of the C-terminal domain to further analyze its karyophilic determinants. Plasmids expressing these mutants fused to maltose binding protein (MBP) and enhanced green fluorescent protein (EGFP) were transfected to COS-1 cells and subcellular localization of these fluorescent fusion proteins was determined by fluorescent microscopy. The results revealed that a maximum karyophilicity was exhibited by a region longer than the previously described one of 29 aa (aa 306-334), in particular by a 64 aa region (aa 289-352) with Arg341 and Lys349 as critical determinants.