In parallel with their well-characterized delayed genomic effects, steroid hormones exhibit rapid, non-genomic effects at molecular, cellular and behavioral levels. We have proposed a model of rapid, non-genomic glucocorticoid inhibition of hypothalamic neuroendocrine cells through a putative membrane-associated glucocorticoid receptor (GR). Here we tested for plasma membrane GR immunoreactivity and binding in the hypothalamic supraoptic and paraventricular nuclei. Selective cross-linking of membrane proteins with membrane-impermeant BS3 and subsequent Western blot analysis with a monoclonal GR antibody revealed a reduction in the intensities of a ˜98 kDa immunoreactive band and a ˜64 kDa band in the rat paraventricular and supraoptic nuclei, and of a 64 kDa band in hippocampal tissue, which suggested that these proteins are associated with the membrane. Saturation binding of [3H]-corticosterone and [3H]-dexamethasone in rat and mouse hypothalamic tissue revealed a Kd 4–24-fold lower and a Bmax 4–7-fold lower for the membrane-associated GR compared to the intracellular GR, suggesting a lower affinity and abundance of the glucocorticoid binding sites in the membrane than in the cytosol. Together, these findings suggest the presence of a low-affinity, low-abundance membrane-associated GR in the hypothalamus that shares homology with the intracellular GR, and are consistent with physiological evidence of rapid, non-genomic glucocorticoid actions in hypothalamic neuroendocrine cells that are GR dependent.