The honeybee forms a long-term memory in different training situations that lasts for a lifetime, but the cellular mechanisms of long-term memory formation are not known. We analyzed the dependency of long-term memory on the de novo brain protein synthesis. The protein synthesis inhibitor cycloheximide was injected via the median ocellus directly into the brain. 3H-leucine incorporation into brain proteins was inhibited by > 95% for > 3 hr. The time of protein synthesis inhibition was prolonged by a second injection of the same dose. Worker honeybees were conditioned to an olfactory stimulus at different times before and after injection. The proboscis extension response (PER) of bees restrained in tubes was classically conditioned with sugar water applied first to the antennae followed by feeding (unconditioned stimulus) paired with odor presentation (conditioned stimulus). The bees were tested by presenting the odor alone at different times up to 24 hr after injection. No significant reduction in the probability of the conditioned response in cycloheximide-treated bees was found when compared to the Ringer-injected controls in 4 series of experiments. Since protein synthesis was inhibited between 7 hr pre- and 7 hr postconditioning without affecting the formation of long-term memory, a possible role of de novo protein synthesis in the formation of long- term memory after olfactory conditioning of the PER is not supported by these experiments.