Ghrelin is a 28-amino acid peptide secreted mainly by the stomach. Acyl-ghrelin, which binds to and activates the growth hormone secretagogue receptor type 1a (GHS-R1a), is considered to be the active form for its orexigenic effects. It has been demonstrated that peripheral administration of ghrelin stimulates food intake and adiposity in rodents and humans. Accordingly, different approaches to antagonize ghrelin/GHS-R1a signaling have been pursued for the treatment of obesity. In the present study, we generated and characterized high-affinity anti-acyl ghrelin-specific monoclonal antibodies (mAbs). In vitro, the lead mAb (33A) displayed specific binding to acyl-ghrelin, with an estimated K(d) value < 100 pM. In recombinant receptor cell-based assays, 33A dose-dependently inhibited the ghrelin-mediated calcium signal, with an IC(50) of approximately 3.5 nM. In vivo, ghrelin dose-dependently stimulated food intake in mice, and this effect was fully blocked by a single injection of 33A. In a 4-week chronic study, 33A was shown to effectively bind to endogenous acyl-ghrelin; however, long-term administration of 33A did not affect food intake or body weight gain in a mouse model of diet-induced obesity. Our results indicate that peripheral neutralization of ghrelin can suppress appetite stimulated by a transient surge in ghrelin levels. The lack of long-term effects on body weight control by 33A suggests that compensatory mechanisms may contribute to the regulation of energy balance.