The maturation of the neurosecretory activity of a hypothalamic nerve cell population grown in vitro, prepared from 10-day-old mice and cultured for 6 days, has been demonstrated in the present report. A low-molecular weight polypeptide of 30-kD was found to be released into the culture media during the 6-day period of incubation, as analyzed by SDS-polyacrylamide gel electrophoresis. Comparative electrophoresis of the in situ hypothalamic, neurohypophysis and cerebral cortex homogenates revealed the presence of a 30-kD protein component in both the hypothalamus and neurohypophysis but not in the cerebral cortex. The release of the 30-kD polypeptide into the incubation media indicates an expression of the neurosecretory activity of the peptidergic neurons of the hypothalamus during in vitro maturation. On the other hand, high pressure liquid chromatography with electrochemical detection showed appreciable quantities of released dopamine (DA), epinephrine and serotonin (5-HT) in the incubation media in which the neurons were allowed to differentiate. There was a steady release of DA during the 6-day incubation period, varying from 0.21 +/- 0.02 to 0.49 +/- 0.05 ng/mg protein. The epinephrine level increased progressively from day 1 to 6 of culture, from 3.73 +/- 0.57 to 12.08 +/- 1.81 ng/mg protein, respectively. The measured 5-HT level was 0.07 +/- 0.001 on day 2 and increased to 0.38 +/- 0.05 ng/mg protein on day 6 of culture. These data demonstrate the functional maturation of catecholaminergic, serotoninergic and peptidergic neurons in these rotary histotypic cultures of the mouse hypothalamus.