Cart (cocaine- and amphetamine-regulated transcript) was first identified to be a major brain mRNA up-regulated by cocaine and amphetamine. The CART protein has been established as a satiety factor closely associated with the action of leptin. To assess CART’s role as an anorexigenic signal, we have generated CART-deficient mice by gene targeting. On a high fat diet, CART-deficient and female heterozygous mice, but not male heterozygous mice, showed statistically significant increases in weekly food consumption, body weight, and fat mass compared with their wild-type littermates. Furthermore, CART-deficient and female heterozygous mice were significantly heavier when fed a high fat diet than on a regular chow diet at 17 wk of age and at the 14th wk of the feeding studies. However, wild-type or male heterozygous mice showed no weight variations attributable to caloric contents of the diet at that age. Contrary to the obese phenotypes shown in MC4R-, proopiomelanocortin-, or leptin-deficient mice, our results showed that CART deficiency predisposed mice to become obese on a calorically dense diet. The results also show that CART may not be a major anorectic signal compared with proopiomelanocortin or leptin in the regulation of energy homeostasis.