Oscillations in intracellular calcium levels have been described in GnRH-1 neurons in both prenatal and adult cells. However, differences have been reported in the mechanisms underlying these [Ca(2+)](i) oscillations, dependent on the model used. The goal of this study was to address whether these changes depend on the maturation status of GnRH-1 neurons by assaying prenatal GnRH-1 cells maintained in explants, at two different developmental stages. This report documents an increase in the frequency of [Ca(2+)](i) oscillations between 1 and 3 wk of in vitro maturation. During the early stage, [Ca(2+)](i) oscillations are blocked by tetrodotoxin and are mainly triggered by excitatory neurotransmitters, gamma-aminobutyric acid (GABA), and glutamate. In contrast, in the later stage, some cells exhibit residual tetrodotoxin-insensitive [Ca(2+)](i) oscillations, which are sustained by action potential-independent GABA and glutamate release. The strength of these two excitatory inputs remained relatively constant during the maturation process, and the increase in frequency of [Ca(2+)](i) oscillations observed at the later stage is due to a novel excitatory input carried by cholecystokinin. Together, these data indicate developmentally regulated release and interactions of neurotransmitters (known regulators of GnRH-1 cells in adults) and point to extrinsic factors regulating GnRH-1 cellular physiology.