The aim of the present study was to test the hypothesis that there should be a difference between the effects of an acute and an 8‐day (chronic) administration of fluoxetine (10 mg/kg) on the rate of serotonin [5‐hydroxytryptamine (5‐HT)] synthesis. The 5‐HT synthesis rate was measured in discrete regions of the rat brain using the α‐[14C]methyl‐l‐tryptophan autoradiographic method. The results show that the acute and chronic fluoxetine treatments influence the 5‐HT synthesis rate in different ways. A single dose of fluoxetine induced a significant increase in 5‐HT synthesis in the visual, auditory, and parietal cortices, substantia nigra, hypothalamus, ventral thalamus, and dorsal hippocampus. In contrast, after a chronic treatment a decrease was observed in the substantia nigra, caudate, and nucleus accumbens, the auditory, parietal, sensorimotor, and frontal cortices, and ventral tegmental area. A significant decrease in the rate of 5‐HT synthesis was observed in the dorsal raphe after both the single and chronic treatments. The results suggest that extracellular 5‐HT has a delayed influence on the brain 5‐HT synthesis rate in structures with serotonergic terminals. The findings from the acute study could be important for patients who have just started receiving fluoxetine treatment, as an increase in the 5‐HT synthesis rate might occur in the acute phase of their treatment. In addition, the findings from the chronic treatment study might give us a better understanding of how the brain serotonergic system adapts during a prolonged exposure to extracellular 5‐HT.