The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus for regulating homeostatic, neuroendocrine, and behavioral functions. We conducted immunocytochemical analyses by using antisera directed against gamma-aminobutyric acid (GABA), its synthetic enzyme glutamic acid decarboxylase (GAD67), GABA-A receptor subunits (alpha2, beta3, epsilon), estrogen receptor-alpha, and Neuropeptide Y (NPY) in the region of the VMH in embryonic mice to identify potential patterning elements for VMH formation. Cells and fibers containing GABA and GAD67 encircled the primordial VMH as early as embryonic day 13 (E13) when the cytoarchitecture of the VMH was not recognizable by Nissl stain. At E16-17 the cytoarchitecture of the VMH became recognizable by Nissl stain as GABAergic fibers invaded the nucleus, continued postnatally, and by adulthood the density of GABAergic fibers was greater inside than outside the VMH. GABA-A receptor subunit expression (beta3 by E13 and alpha2 by E15) within the primordial VMH suggested potential sensitivity to the surrounding GABA signal. Brain slices were used to test whether fibers from distal or proximal sites influenced VMH development. Coronal Vibratome slices were prepared and maintained in vitro for 0-3 days. Nissl stain analyses showed a uniform distribution of cells in the region of the VMH on the day of plating (E15). After 3 days in vitro, cellular aggregation suggesting VMH formation was seen. Nuclear formation in vitro suggests that key factors resided locally within the coronal plane of the slices. It is suggested that either GABA intrinsic to the region nearby the VMH directly influences the development and organization of the VMH, or along with other markers provides an early indicator of pattern determination that precedes the cellular organization of the VMH.