We studied the effects of Zn(2+) on human brain creatine kinase (HBCK). Zn(2+) inactivated the activity of HBCK in a dose dependent manner (IC50 = 0.06 mM). The time-interval kinetic studies showed that the inactivation followed first-order reaction kinetics with a biphasic process. The spectroflurorimetry results showed that Zn(2+) conspicuously induced the tertiary structural change of HBCK with exposure of its hydrophobic surfaces. On the contrary, the secondary structure was slightly changed by Zn(2+). We also found that HBCK aggregation was induced by Zn(2+). This aggregation was dependent on the temperature and the enzyme and Zn(2+) concentrations. Some added osmolytes such as glycine and proline were able to successfully block CK aggregation and fully recover the conformation and activity of HBCK. Our study provides important insight into the unfavorable effect of Zn(2+) on HBCK and it increases the understanding of the Zn(2+) ligand-binding mechanism to the metabolic brain enzyme.