The adipose-derived hormone leptin communicates information about metabolic status to the hypothalamic GnRH neuronal system. It is unclear whether leptin can act directly on GnRH neurons. To examine this, we used three approaches. First, the presence of leptin-induced signal transducer and activator of transcription-3 activation was examined in GnRH neurons in male and female rats. Intracerebroventricular treatment with 4 mug leptin-induced robust signal transducer and activator of transcription-3 expression within the anteroventral periventricular nucleus but not in GnRH neurons. Second, fertility was assessed in male and female CRE-loxP transgenic mice with conditional leptin receptor (Lepr) deletion from either all forebrain neurons or GnRH neurons only. Forebrain neuron LEPR deletion prevented the onset of puberty resulting in infertility in males and females and blocked estradiol-induced LH surge. However, mice with GnRH neuron-selective Lepr deletion exhibited normal fertility apart from a slight puberty delay in males. Lastly, the highly sensitive technique of single-cell nested PCR was used to test for Lepr transcript presence in individual GnRH neurons, identified in situ using GnRH-green fluorescent protein transgenics. Whereas 75% of positive control (proopiomelanocortin) neurons contained Lepr mRNA, no (none of 18) GnRH neurons were Lepr mRNA positive. Collectively, these results show that leptin does not act directly on GnRH neurons in rats and mice. Leptin appears to regulate GnRH function via forebrain neurons that are afferent to GnRH because forebrain neuronal LEPR deletion caused infertility. The location and phenotype of these leptin-responsive neurons remains to be elucidated.