The purpose of the present study was to investigate the effect of phenobarbital on some brain and liver tissue enzyme activities in Balb/C mice. Forty male Balb/C mice were used. Ten mice served as a control group, and thirty mice were administered with phenobarbital (80 mg·kg-1 body mass, single oral dose). Brain and liver samples were taken at 6, 12 and 24 h after drug administration. Brain and liver tissues alkaline phosphatase, aspartate aminotransferase, gamma glutamyl transferase and amylase activities were measured by autoanalyzer. Phenobarbital did not affect gamma glutamyl transferase and amylase activities in the brain and liver, and alkaline phosphatase and aspartate aminotransferase activities in the liver. However, statistically significant (p < 0.05) increases of alkaline phosphatase and aspartate aminotransferase activities were observed in the brain. In general, after phenobarbital administration, serum alkaline phosphatase and aspartate aminotransferase activities increase and these increases are believed to be derived from the liver. These result suggest that brain may contribute to increased activities of these enzymes in serum in Balb/C mice.