The hypothalamic arcuate nucleus (Arc) is the presumed target site for the orexigenic hormone ghrelin which is secreted from the stomach during fasting. Ghrelin directly activates Arc neurones. Similar to exogenous ghrelin, overnight food deprivation also induces c-Fos expression in the Arc of mice. In this study we tested the role of endogenous ghrelin in the fasting-induced c-Fos expression in the Arc of mice. We used NOX-B11-3, the latest generation of the recently developed ghrelin-binding compounds, so-called RNA Spiegelmers (SPM) to block endogenous ghrelin action during food deprivation. The specificity and potency of this compound was also tested in electrophysiological and immunohistological experiments. In electrophysiological in vitro single cell recordings NOX-B11-3 effectively blocked the excitatory effect of ghrelin in the medial Arc (ArcM) of rats while the biologically inactive control SPM had no effect. Furthermore, NOX-B11-3 (15 mg/kg ip) potently suppressed ghrelin-induced (25 µg/kg sc, 12h after SPM injection) c-Fos expression in the Arc. However, the same dose of NOX-B11-3 when injected at the beginning of a 14h fasting period had no effect on the c-Fos expression in the Arc of mice. These results demonstrate that NOX-B11-3 is a long acting compound, which effectively blocks the effect of exogenous ghrelin on neuronal activity in the Arc under in vitro and in vivo conditions. Furthermore, increased ghrelin signalling does not appear to be a necessary factor for the activation of Arc neurones during food deprivation or other fasting-related signals might have masked or compensated the loss of the ghrelin effect.