Mice deficient in neurogenin 3 (Ngn3) fail to generate pancreatic endocrine cells and intestinal endocrine cells. Hypothalamic neuropeptides implicated in the control of energy homeostasis might also be affected in Ngn3 homozygous null mutant mice. We investigated the expression of two prominent orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), in the hypothalamic arcuate nucleus of newborn wild-type and Ngn3 null mutant mice. Immunohistochemical analysis demonstrated that, in Ngn3 null mutants, the number of NPY-immunoreactive neurons and nerve fibers was markedly increased in the arcuate nucleus, and the nerve fibers were widely distributed in the hypothalamic area, including the paraventricular and dorsomedial nuclei. Little increase of AgRP immunoreactivity was detected in the arcuate nucleus of mutant mice. In situ hybridization analysis confirmed the increased population of the NPY neurons in the arcuate nucleus of the mutants. The NPY mRNA level, as estimated by laser capture microdissection and real-time quantitative polymerase chain reaction, was 371% higher in Ngn3 null mutants than in wild-type mice. AgRP mRNA levels did not differ significantly between the null mutants and wild-type mice. Thus, up-regulation of the hypothalamic NPY system is probably a feature characteristic of Ngn3 null mice.