Some of the side effects that develop during long-term valproic acid (VPA) treatment are similar to symptoms of zinc and partial biotinidase deficiencies. This situation suggests an association between these side effects and biotinidase and/or zinc deficiencies. In 32 pediatric patients (22 females, 10 males) receiving VPA treatment, hair and serum zinc levels and serum biotinidase activity (BA) were measured prior to and in the 3rd and 6th months of treatment. Also, serum VPA levels were measured in the 3rd and 6th months of treatment. The mean serum and hair zinc levels were found to be reduced in the 3rd and 6th months of treatment as compared with the pre-treatment values, while the mean serum BA was lower than the pre-treatment values in the 3rd month of treatment, but returned to initial values in the 6th month of treatment. In the 3rd and 6th months of treatment, patients complaining about hair loss had lower hair and serum zinc levels and serum BA but greater mean serum VPA than those who did not. However, the differences between parameters were not statistically significant. Our findings suggest that hair loss in patients can be attributed to zinc and BA depletion within the first 3 months, and to zinc depletion only by the 6th month.