Nonhistone nuclear proteins were isolated from 3-5 day old neonatal as well as 3 month-old adult myocardium. The nuclear proteins were separated and analyzed by two-dimensional polyacrylamide gel electrophoresis. Using a blot transfer technique equilibrated with 65Zn2+, at least four polypeptides exhibited Zn(2+)-binding activity over the spectrum of nonhistone nuclear proteins. A protein with a molecular weight of 68kDa pI7.8, which has been characterized for its involvement in nucleosome structure, consistently binds Zn2+ in both the neonatal and adult myocardium. This nuclear protein has now been further characterized by partial amino acid microsequencing. It was found that this novel polypeptide is distinct from the pore-complex lamina proteins. Three other polypeptides with M tau 90kDa, pI7.8, M tau 68kDa, pI6.5 and M tau 35kDa, pI7.5 exhibited increased Zn(2+)-binding activity in neonatal myocardium as compared to adult myocardium. Together with results from our previous studies, this study provides the first evidence implicating Zn(++)-binding nuclear proteins in the processes of growth and differentiation of myocardial development.