The effect of actinomycin D (ACTD) at three dosage levels on the enzymatic activities of the normal rat pancreas was studied. At the 1.0 and 0.5 mug/g total body weight dosages there were decreased levels of amylase, lipase and chymotrypsin 1 hour after injection, a return to control levels shortly thereafter and a second drop in enzymatic levels a day or two later. Since ACTD is known to affect transcription of DNA within hours of administration, the first decrease may have reflected the effect of ACTD on the synthesis of enzyme mRNA. The lowest dosage, 0.05 mug/g, said to affect the synthesis of rRNA only, gave decreased enzymatic activities from 1-3 days after injection, suggesting that rRNA synthesis was involved, primarily. There were variable effects of ACTD on the enzymatic levels of regenerating pancreas after ethionine. When given for a single day early in the regeneration period, the injection of the 0.5 mug/g dosage, believed to affect both mRNA and rRNA synthesis, decreased considerably the levels of enzymatic activity on the fifth day of regeneration. Injection on multiple day early in regeneration, however, gave a paradoxic effect-higher levels than control values. The lowest dosage, 0.05 mug/g, affecting primarily rRNA, caused, even with multiple doses, a considerable decrease of enzymatic values to well below control levels. The paradoxic effect of the higher concentration of ACTD when given on multiple days may have been related to a decreased synthesis of the repressor of the mRNA for the enzyme, thereby making more mRNA available for synthesis of the enzyme.