Removal of protamine from DNA-protamine (salmine, protamine from salmon sperm) complexes by nucleoplasmin was examined and compared with that of poly-L-glutamic acid (PLGA) using turbidity and ethidium bromide (EB) treatment methods. When nucleoplasmin or PLGA was added to a DNA-protamine complex solution, turbidity was decreased and the amount of EB intercalated into DNA was increased. These results suggest that nucleoplasmin and PLGA can remove protamine from DNA-protamine complexes. The effect of nucleoplasmin was more potent than that of PLGA. Direct interaction of nucleoplasmin with protamine was confirmed by mixing experiments using circular dichroism (CD) and fluorescence spectroscopies. Results suggest that nucleoplasmin is bound to protamine in a 1:1 ratio and that Trp126 is located near a hydrophilic region containing a polyglutamic acid tract of nucleoplasmin which was obviously influenced by its binding with protamine. It would appear that the polyglutamic acid tract in nucleoplasmin plays a critical role for binding with protamine.