The drug malononitrile dimer (1,1,3-tricyano-2-amino-1-propene) was administered intraperitoneally to groups of mice at 20 mg/kg body weight, either as a single injection or as chronic 2-month injections. To study the effect of malononitrile dimer on RNA synthesis in the brain, it was necessary to choose a precursor that would pass the blood-brain barrier. Animals were injected with 14C-formate, then sacrificed 1 h later. RNA synthesis in the brain, liver and kidney of the drug-injected and control animals was determined by the amount of 14C-formate incorporated into newly synthesized RNA. A new method involving the comparison of RNase-extracted with non-extracted slides is described for determining the proportion of 14C-formate incorporated into RNA radioautographically. The results show that RNA synthesis was significantly increased as a result of both single and chronic malononitrile dimer injections in all tissues studied. In most cases, malononitrile dimer nearly doubled the percentage of 14C-formate incorporated into newly synthesized RNA. The effect of the chronic injections was most pronounced in the brain. The mechanism of malononitrile action on RNA synthesis are mediated by thyroxine is discussed.