The progestin 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) facilitates sexual receptivity when applied to the midbrain ventral tegmental area (VTA) of rodents. The concentration of 3alpha,5alpha-THP in the midbrain was investigated in hormonal states associated with hormonally-dependent changes in behavior, notably increases in sexual receptivity in rodents. Rats and hamsters were ovariectomized, treated with vehicle, estradiol benzoate (EB), or EB+progesterone (EB+P). 3alpha,5alpha-THP was measured by radioimmunoassay in plasma, whole brain, cortex, hippocampus, hypothalamus, midbrain, and amygdala. 3alpha,5alpha-THP was further localized in cortex, CA1 region of the hippocampus, ventral medial hypothalamus, VTA, and dorsolateral amygdala by immunocytochemistry (ICC). 3alpha,5alpha-THP concentrations were highest in cortex and hippocampus. Midbrain concentrations of 3alpha,5alpha-THP were greater than in the hypothalamus or amygdala. In rats, 3alpha,5alpha-THP was increased after EB and further elevated following P administration. In hamsters, similar levels of 3alpha,5alpha-THP were seen after EB or EB+P. Overall, 3alpha,5alpha-THP was increased in the midbrain VTA of rats and hamsters that were administered hormone regimens that are associated with estrous variations and enhancement of sexual receptivity in rats and hamsters.