The time course of incorporation and cellular localization of 3H-5-hydroxytryptophan (3H-5-HTP) and 3H-5-hydroxytryptamine (3H-5-HT) in the pineal and some brain regions in rainbow trout, Salmo gairdneri, were studied by quantitative and qualitative autoradiography. Among the tissues examined, the pineal shows the highest and most rapid uptake of the two isotopes. Maximum incorporation of 3H-5-HTP is achieved by 2 h and that of 3H-5-HT by 20 minutes post injection. At the end of the six-hour experimental period, a significantly high amount of radioactivity is still detectable in the pineal. The results indicate a much slower turnover of the two indoles, especially 5-HTP, in the trout than is known for mammalian tissues. Both the ependymal supporting cells and the receptor cells of the pineal localize these isotopes. In constant, the intrapienal neurons remain unlabeled. This is taken to suggest lack of capacity of these cells to metabolize 5-HTP and 5-HT. In the circumventricular regions, the two indoles occur in the ependyma of the recessus lateralis and the recessus praeopticus. The label is also localized in the neuropil and the neurons of the nuclei recessus lateralis and praeopticus. Semiquantitative estimates reveal a significant labeling of these areas only 20 minutes after injection, although a weak but inconsistent labeling of the ependyma is evident at 5 minutes. The significance of these results is discussed in regard to (a) normal capacity of circumventricular areas to metabolize indoles and (b) a possible chemical interaction between the pineal and the brain involving a direct pineal-cerebrospinal fluid pathway.