Ghrelin and the ghrelin receptor (growth hormone secretagogue receptor, GHS-R), are believed to have important roles in energy homeostasis. We describe results from the first studies to be conducted in congenic (N10) adult ghrelin-/- and Ghsr-/- mice under conditions of both positive (high fat diet) and negative (caloric restriction) energy balance. In contrast to results from young N2 mutant mice, changes in body weight and energy expenditure are not clearly distinguishable across genotypes. Although RQ was lower in mice fed a high fat diet, no differences were evident between littermate wild-type (WT) and null genotypes. With normal chow, a modest decrease trend in respiratory quotient (RQ) was detected in ghrelin-/- mice, but not in Ghsr-/- mice. Under caloric restriction, the weight loss of ghrelin-/- and Ghsr-/- mice was identical to WT littermates, but blood glucose levels were significantly lower. We conclude that adult congenic ghrelin-/- and Ghsr-/- mice are not resistant to diet-induced obesity, but under conditions of negative energy balance show impairment in maintaining glucose homeostasis. These results support our hypothesis that the primary metabolic function of ghrelin in adult mice is to modulate glucose sensing and insulin sensitivity, rather than directly regulate energy intake and energy expenditure.