Thiopental-³⁵S having a specific radioactivity of 13.2 mCi/mmole was synthesized from H₂³⁵SO₄ as the starting material through H₂³⁵S and thiourea-³⁵S. Radiochemical and chemical yields from H₂³⁵SO₄ were 80.3 and 85.7%, respectively. The isotope dilution analysis for the determination of commercial thiopental sodium preparations was investigated with the ³⁵S-labeled thiopental, and it gave the mean recovery of 99.60±0.68%. This method was advantageous over other known official methods such as ultraviolet absorptiometry and gravimetric analysis in the precision and selectivity of the method. As the metabolites of thiopental in the liver of male rats, methionine, thiourea, inorganic sulfate, and several unidentified ³⁵S-containing compounds were recognized by radiopaperscannography or reverse isotope dilution method. Metabolic rates of thiopental-³⁵S in rat liver were 0, 49.7, 59.2, and 60.2% at 15, 30, 90, and 120 minutes after its intraperitoneal injection, respectively. It was proved that a considerable amount of radioactivities arising from the injected ³⁵S-labeled thiopental was distributed in rat brain within 15 minutes after its intraperitoneal adminstration, and that the rapid reduction in radioactivities was found to three-fifths of the original during the following 15 minutes.