The expression of messenger RNAs encoding for tryptophan hydroxylase (TPOH), the first enzyme involved in serotonin and melatonin synthesis, has been investigated by in situ hybridization during the development of the rat pineal gland. TPOH mRNAs were detected as early as the twentieth day of gestation (E20) in the rat embryo before any nerve ending was observed in the pineal gland. After birth, their expression increased strongly, and attained a plateau during the second week. This coincides with the setting up of sympathetic innervation. From day 17 (D17), the TPOH mRNA expression diminished. These results indicate that noradrenergic innervation is not involved in the initiation of rat pinealocyte differentiation, but might modulate cell maturation. This study showed the existence of three types of cells arranged in patches in the young rat pineal gland (D6): regions in which cells expressed TPOH mRNAs, regions in which cells expressed vimentin, an intermediate filament protein present in the cytoskeleton of immature cells, and regions in which both TPOH mRNAs and vimentin are expressed. In older rat pineal gland (D20), almost all cells express TPOH mRNAs, and some cells still express vimentin. This suggests that all cells do not reach the same level of differentiation at the same time in the rat pineal gland.