The distribution of GABAA receptor sites was studied in female and male rat brain by an improved in vitro binding assay using 3H-muscimol and carefully washed membrane fractions. The binding studies revealed a single class of binding sites exhibiting the properties of GABAA receptors. The specific binding of 3H-muscimol was measured in four brain areas: the preoptic brain area, mediobasal hypothalamus, corticomedial amygdala and cerebral cortex. The equilibrium dissociation constants (KD) ranged from 11.2 to 23.3 nM in diestrous females and from 13.1 to 50.4 nM in males, the maximal number of binding sites (Bmax) from 3290 to 10240 fmol/mg protein in females and from 5495 to 17449 fmol/mg protein in males. In the preoptic area and mediobasal hypothalamus both, KD and Bmax, were significantly higher in males than in females. The sex differences in the binding of muscimol observed in vitro indicated sexually dimorphic GABAergic neurotransmission that is probably related to the control of gonadotropin secretion and/or sexual behavior.