Genetic instruction is one of the principal aspects of a body plan including the neural network formation. In the olfactory receptor neurons (ORNs) as well, odorant receptor genes are a key determinant of the axonal targeting to the glomeruli in the olfactory bulb to form the network called the olfactory map. Interestingly, ORNs periodically regenerate and also utilize spontaneous firing activity for establishing and maintaining this olfactory neural map. However, (1) the generation mechanism of spontaneous activity and (2) the its role as a guidance cue in the olfactory bulb are not established. Here we addressed these two questions by investigating the nature of the spontaneous ORN firing activity in the acute slice preparations of mouse olfactory epithelium. We detected the predominant immunoreactivities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subtypes 2 and 4 in soma of ORNs. By applying extracellular recording, we found that HCN channels depolarized the membrane of ORNs and boosted the spontaneous firing activity by sensing the cAMP levels that was largely maintained by the basal activation of Gs-coupled beta2-adrenergic receptor; the basal activity of beta2-adrenergic receptor was inhibited by ICI 118,551 (IC50 = 4 µM). We also demonstrated by a whole-cell voltage-clamp configuration that the activation of the olfactory HCN channels (control: V1/2 = -115.7 ± 3.5 mV) was positive-shifted by 1 µM intracellular PIP2 (V1/2 = -98.2 ± 4.6 mV) and further by 1 mM 8-Br-cAMP (V1/2 = -86.0 ± 1.5 mV). The results indicate that HCN channels can be operational at the resting membrane potentials of ORNs (about -70 to -90 mV). Furthermore, we generated mice with HCN4 channel over-expressed by Tet-system, in which the spontaneous ORN firing rate was much higher than that in non-transgenic littermates. Strikingly, the histological architecture of the olfactory map was severely deteriorated; the olfactory bulb became much smaller and the size and number of the olfactory glomeruli were drastically reduced especially in the dorsal region (the maximum numbers of glomeruli was 42.5 ± 1.8 glomeruli in control mice and 23.1 ± 0.5 glomeruli in HCN4 over-expressing mice). Finally, we confirmed that the knock-down of the over-expressed HCN4 by tetracycline derivative successfully recovered these structural changes. Thus we conclude the rate of the spontaneous electrical activity is a unique guidance cue intrinsically regulated within an optimal range to maximize the diversity and integrity of the olfactory map (Nakashima et al., 2013).