The biosynthesis of RNA in the cultured rat pineal was monitored during the (l)-isoproterenol-induced increase in N-acetyltransferase activity. Treatment with the β-agonist (l)-isoproterenol elevated pineal N-acetyltransferase activity 30-fold within 6 hr but had no effect on the incorporation of [3H]uridine into total nuclear, total cytoplasmic or total poly(A) containing RNA. Examination of the [3H]uridine-labeled total cytoplasmic RNA and poly(A)-rich messenger RNA by means of agarose gel electrophoresis revealed no significant changes in the synthesis of these RNA species by (l)-isoproterenol treatment of the cultured pineal gland. The lag in the appearance of newly synthesized RNA from the nucleus into the cytosol of the pineal closely corresponds to the lag in the appearance of new N-acetyltransferase activity. These results suggest that, although (l)-isoproterenol may cause a dramatic induction of N-acetyltransferase enzyme activity in the cultured pineal gland, there is no significant alteration in the RNA biosynthesis of the pineal gland to meet the demands of enzyme induction. The induction of N-acetyltransferase activity by (l)-isoproterenol is inhibited by actinomycin D. However, sensitivity to the antibiotic is lost 90-180 min after the initiation of induction by (l)-isoproterenol but 90 min before the accumulation of appreciable enzyme activity.