The involvement of new protein and messenger ribonucleic acid synthesis in long-term potentiation was studied in the anaesthetized rat dentate gyrus using several inhibitors of protein synthesis (anisomycin, emetine, cycloheximide and puromycin) and an inhibitor of messenger ribonucleic acid synthesis (actinomycin D). When injected for 1 h just prior to tetanization, the four inhibitors of protein synthesis produced a mild reduction of long-term potentiation of the excitatory postsynaptic potential measured 10 min after tetanization. Anisomycin produced a significantly faster decay of long-term potentiation, while the other inhibitors had more moderate effects. Actinomycin D failed to affect long-term potentiation. In a second experiment, the time-dependency of the anisomycin effect was examined. Anisomycin injected immediately after tetanization promoted decay of long-term potentiation, but when injected after a 15-min delay, the drug had no effect. Inhibition of protein synthesis for 4 h prior to tetanization did not have any more effect on long-term potentiation than inhibition for 1 h. In no experiment was long-term potentiation of the population spike affected by drug manipulation. These results suggest that for long-term potentiation of the excitatory postsynaptic potential to be maintained for at least 3 h proteins must be synthesized from already existing messenger ribonucleic acid, and that this synthesis is mostly completed within 15 min after tetanization.