1. As shown by a double-radioisotope technique in vivo, at a non-lethal dose of cycloheximide, a stimulation of nuclear RNA synthesis occurred by 12 h after the treatment; the stimulation lasted over 48 h. Analysis of radioactive nuclear RNA by gel electrophoresis demonstrated that most of the cycloheximide-stimulated synthesis could be accounted for by known rRNA precursors (45 S, 41 S, 32 S and 28 S). 2. During the inhibitory phase of protein synthesis, 2 h after cycloheximide treatment, synthesis of the poly(A)-containing mRNA isolated from the cytoplasmic ribonucleoprotein complexes with an oligo(dT)-cellulose column was stimulated, whereas the synthesis of rRNA was slightly inhibited. However, during the stimulatory phase of protein synthesis, 24 h after cycloheximide treatment, the syntheses of both poly(A)-containing mRNA and rRNA were enhanced. 3. Kinetic studies revealed that the newly synthesized RNA species were transported from the nuclei, integrated into the ribonucleoprotein complexes, and associated with both free and membrane-bound polyribosomes. 4. These data corroborate our proposal that the stimulated protein synthesis after cycloheximide administration involves gene transcription.