We studied the effect of Zn2+ on the folding and aggregation of brain creatine kinase (CK-BB). We developed a method to purify CK-BB from rabbit brain and conducted inhibition kinetics and unfolding studies of CK-BB. Zn2+ conspicuously aggregated and osmolytes, such as glycine and proline, were able to suppress the formation of aggregates and protect the enzymatic activity against Zn2+. These results suggest that Zn2+ might act as a risk factor for CK-BB in the brain under certain conditions, and some osmolytes may help CK-BB to sustain the active state when Zn2+ is present. Our study provides useful information regarding the effect of Zn2+ on brain-derived metabolic enzymes, especially those that are putatively related to brain disease. Furthermore, our study suggests that although Zn2+ may induce CK-BB inactivation and misfolding, the ability of some abundant proteins and osmolytes to chelate Zn2+ nonspecifically may protect CK-BB and allow it to exist in the active form.