In order to analyze the feedback action of prolactin (PRL) on the hypothalamus on the cellular level, we used primary cultures of rat embryonic diencephalon to measure the calcium response of individual neurons to PRL by means of fast fluorescence photometry. The cultures were subsequently stained with antibodies against the neuronal marker MAP-2, glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH). PRL caused a rapid rise of intracellular free Ca2+ in a specific type of GABAergic neuron characterized by a spindle-shaped bipolar morphology and immunoreactivity to MAP-2 and GAD but not to TH. It is concluded that a subpopulation of hypothalamic GABAergic but not dopaminergic neurons react to PRL with a rapid increase in intracellular free Ca2+. These data are compatible with the assumption of a rapid negative feedback regulation of the secretion of PRL from the pituitary mediated by tuberoinfundibular GABAergic neurons.