This study used in situ hybridization (ISH) to examine the distribution of estrogen receptor beta (ERbeta) mRNA in hypothalamic, limbic, and midbrain regions of monkey brain and its regulation by estrogen (E) and progesterone (P). Monkey-specific ERbeta cDNAs were developed with human primers and reverse transcription and polymerase chain reaction (RT-PCR) using mRNA extracted from a rhesus monkey prostate gland. ERbeta 5' (262 bases) and 3' (205 bases) riboprobes were used in combination for ISH. Ovariectomized and hysterectomized (spayed) pigtail macaques (Macaca nemestrina; four per treatment group) were either untreated spayed-controls, treated with E (28 days), or treated with E plus P (14 days E+14 days E and P). Dense ERbeta hybridization signal was seen in the preoptic area, paraventricular nucleus, and ventromedial nucleus of the hypothalamus; the substantia nigra, caudal linear, dorsal raphe, and pontine nuclei of the midbrain; the dentate gyrus, CA1, CA2, CA3, CA4, and the prosubiculum/subiculum areas of the hippocampus. Expression in the suprachiasmatic region, supraoptic nucleus, arcuate nucleus, and amygdala was less intense. Image analysis of the dense areas showed no significant difference in the hybridization signal in individual regions of the hypothalamus, midbrain, or hippocampus between any of the treatment groups. However, P treatment decreased overall ERbeta signal in the hypothalamus and hippocampus when several different subregions were combined. The localization of ERbeta in monkey brain by ISH is in general agreement with that previously described in rodents. The presence of monkey ERbeta mRNA in brain regions that lack ERalpha should help to clarify the molecular mechanisms by which E acts in the central nervous system to influence hormone secretion, mood disorders, cognition, and neuroprotection.