The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 μM and 5 μM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 μM and 100 μM in the pipette, the half-maximal activation voltage (V1/2) was shifted by +28.5±1.7 mV. The activation time constant (τa) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. τa changed from 1106±60 ms at 0 μM cAMP to 265±7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 μM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 μM forskolin induced an elevation of the cAMP level by about 1.6±0.2 μM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4(a)- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4(a)-receptors by 0.1 μM 5-HT induced an increase of cAMP of about 4.6±1.5 μM, whereas cAMP levels decreased from a control value of 1±0.2 μM to 0.41±0.1 μM after stimulation of the m5-HT1A-receptors.