Cultures of endogenous GnRH neurons and the GT1 GnRH neuronal cell line release GnRH in pulses (intrinsic pulsatile release) with an interpulse frequency similar to that seen in castrated animals. Both in GT1 cells and transgenic rats, lowering cAMP levels by expression of a phosphodiesterase decreased the frequency of intrinsic GnRH pulsatility. We asked whether the cAMP-gated cation (CNG) channels, expressed in GT1 cells participated in cAMP modulation of intrinsic GnRH pulsatility. Since expression of the CNGA2 subunit is essential for formation of functional CNG channels, we developed an adenovirus vector expressing a short interference RNA (siRNA), to the CNGA2 subunit (Ad-CNG-siRNA) or as an infection control, to the coding region of luciferase (Ad-Luc-siRNA). Infection with the Ad-CNG-siRNA of COS cells transfected with a CNGA2 expression vector significantly inhibited CNGA2 protein levels by 74% by Western blot. Infection of GT1-1 cells with Ad-CNG-siRNA resulted in a 68% decrease in the levels of CNGA2 mRNA, a 44% decrease in protein levels and a clear decrease in immunostaining with an antibody to CNGA2. Infection of GT1-1 cells with Ad-CNG-siRNA decreased spontaneous Ca²⁺ oscillations compared with Ad-Luc-siRNA infected or uninfected cells, by 71%. Furthermore infection with Ad-CNG-siRNA resulted in a 2-fold increase in the interpulse interval in GnRH secretion (49.4 ± 9.1 min) compared to uninfected cells (25.9 ± 2.5 min) or Ad-Luc-siRNA (29.3 ± 2.8 min) infected cells. These data provide the first direct evidence that the CNG channel is a downstream signaling molecule in the regulation of the frequency of intrinsic GnRH pulsatility by cAMP.