A nuclear localization signal binding protein in nuclear envelope was studied as the first step to determine the mechanism of nuclear protein recognition by nuclear envelope. The rat liver nuclear envelope extract was resolved by SDS-PAGE and ligand blotted with 125I-labeled nucleoplasmin bearing a strong nuclear localization signal. A nuclear localization signal binding protein with molecular mass of 60 kDa (NBP60) was detected in the extract. NBP60 could be extracted with 2% Triton X-100-1 M KCl but not with 1 M KCl, 2 M urea, or 2% Triton X-100. The protein was partitioned to the lower layer in a two phase system using Triton X-114. These results suggested that the protein is an intrinsic membrane protein and has a hydrophobic surface. This protein was bound to not only nucleoplasmin but also the nuclear localization signal peptide of SV 40 large T-antigen (T-peptide) conjugated to human serum albumin. The binding of NBP60 to nucleoplasmin-Sepharose was inhibited by 50% in the presence of 0.12 mM T-peptide. However, a high concentration of 2.1 mM was necessary, when mutant T-peptide in which the essential amino acid lysine was substituted with threonine was used. These results suggested that NBP60 binds specifically to nuclear localization signals. NBP60 extracted from the nuclear envelope was purified by nucleoplasmin-Sepharose affinity chromatography following hydroxyapatite high performance liquid chromatography.