Isolated neurointermediate lobes or neural lobes of the rat pituitary gland attached to the pituitary stalk were incubated in vitro and the spontaneous or electrically (pituitary stalk stimulation, 5 Hz, 1,500 pulses) evoked release of 5-hydroxytryptamine was determined. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe was increased fivefold in the presence of the dopamine receptor antagonist (-)-sulpiride (1 microM). The evoked release of 5-hydroxytryptamine from the isolated neural lobe was not altered by (-)-sulpiride. The evoked release of 5-hydroxytryptamine from the isolated neural lobe in the presence of (-)-sulpiride was less than 5% of that from the combined neurointermediate lobe showing that most of the 5-hydroxytryptamine released from the combined neurointermediate lobe originated in the intermediate lobe. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe in the presence of (-)-sulpiride showed a diurnal variation. It was three to five times higher between 9.30 and 14.00 h than between 8.30 and 9.30 h or between 14.00 and 16.00 h. The 5-hydroxytryptamine tissue content at the end of the incubation experiments also showed similar variations which were, however, less pronounced. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe, in the presence of (-)-sulpiride, was reduced by the preferential GABAA receptor agonist muscimol or the selective GABAB receptor agonist (-)-baclofen in a concentration-dependent manner. Bicuculline, a selective GABAA receptor antagonist inhibited the effect of muscimol, but not that of (-)-baclofen. Bicuculline alone did not affect the release of 5-hydroxytryptamine from the gland. It is concluded that the release of 5-hydroxytryptamine in the intermediate lobe is influenced by both dopaminergic and GABAergic mechanisms.