Pulsatile GnRH secretion is an intrinsic property of GnRH neurons. Since increases in cAMP levels increase excitability and GnRH secretion in the GT1-1 GnRH cell line, we asked whether cAMP levels play a role in timing excitability and intrinsic pulsatile GnRH secretion. The expression of the cAMP-specific phosphodiesterase (PDE4D1) was used in a genetic approach to lower cAMP levels. Cells were infected with an adenovirus vector (Ad) expressing PDE4D1 (PDE-Ad), or for controls with an empty Ad (Null-Ad) or an Ad expressing green fluorescent protein (GFP-Ad). Infection with the PDE-Ad significantly inhibited forskolin-induced increases in cAMP production, GnRH secretion, and Ca2+ oscillations. Infection of GT1-1 cells with the PDE-Ad vs. GFP-Ad or Null-Ad controls significantly decreased spontaneous Ca2+ oscillations and inhibited the frequency of GnRH pulses. These data support the hypothesis that the level of cAMP in GT1 neurons is a component of the biological clock timing neuron excitability and pulsatile GnRH secretion. Genetically targeted expression of PDE4D1 represents a powerful approach to study the role of cAMP levels in specific populations of neurons in transgenic animals.