To investigate the DNA binding activity of the JC virus minor capsid protein, VP2, both wild-type and mutant VP2 were cloned and expressed in Escherichia coli. Southwestern blotting was employed for the DNA-binding assay. The results showed that VP2 was able to bind to DNA, except when either the last 13 or the last 29 amino acids were truncated. The results indicate that the DNA-binding domain of VP2 is located within the last 13 amino acids. Furthermore, we also demonstrated that Lys(332) and Lys(336) within the DNA-binding domain are crucial for DNA binding. The findings may provide further information for understanding the mechanism of virion assembly of the JC virus.