Autoregulatory mechanisms affecting serotonin [5‐hydroxytryptamine (5‐HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5‐HT1A and 5‐HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5‐HT1A and 5‐HT1B receptors in the control of the synthesis and release of [3H]5‐HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5‐HT synthesis and release was observed in the presence of 5‐HT (10(‐8) M), the 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), the 5HT1B/1A agonist 5‐methoxy‐3‐(1,2,5,6‐tetrahydro‐4‐pyridinyl)‐1H‐indole (RU 24969), the 5‐HT1B agonist 3‐(1,2,5,6‐tetrahydropyrid‐4‐yl)pyrrolo[3,2‐b]pyrid‐5‐one (CP‐93,129), and the 5‐HT(1D/1B) agonist sumatriptan. Inhibition of 5‐HT synthesis and release induced by 8‐OH‐DPAT was blocked by chiral N‐tert‐butyl‐3‐[1‐[1‐(2‐methoxy)phenyl]piperazinyl]‐1‐phenylpropionam ide dihydrochloride quaternary‐hydrate (WAY 100135) (10(7) M) or methyl 4‐[4‐[4‐(1,1,3‐trioxo‐2H‐1,2‐benzoisothiazol‐2‐yl)butyl]‐1‐p iperazinyl]‐1Hindole‐2‐carboxylate (SDZ 216‐525) (10(‐7)M), and that of CP‐93,129 was blocked by methiothepin (10(‐7) M). Paradoxically, extracellular levels of [3H]5‐HT increased in the presence of 8‐OH‐DPAT and RU 24969 at 10(‐6) M. 5‐HT uptake experiments showed that these two agonists interacted with the 5‐HT transporter. 5‐HT1 binding sites (620 fmol/mg of protein) and 5‐HT1A (482 fmol/mg of protein) and 5‐HT1B (127 fmol/mg of protein) receptors were detected in 12‐day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5‐HT1A receptors are located on nerve cell bodies, whereas 5‐HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5‐HT1A and 5‐HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.