Recent evidence suggests that two cytochrome P450 aromatase (P450arom) mRNA transcripts are present in the rat brain. One of these contains the entire 5'-coding sequence and correlates with the presence of functional enzyme. We designed a new 255-base pair P450arom probe (AROM255) that recognizes only this full-length P450arom mRNA. Ribonuclease protection assays verified that the cRNA probe synthesized from this construct recognized a single RNA species in brain tissues that express aromatase activity, but not in the cingulate cortex, an area previously shown to contain only the alternate transcript. Moreover, the P450arom mRNA content of the preoptic area was significantly lower in castrates than in intact males or testosterone (T)-treated castrates. We employed 33P-labeled cRNA probes to examine the distribution of P450arom mRNA by in situ hybridization. High levels of mRNA were detected in the medial preoptic nucleus (MPN), bed nucleus of the stria terminalis (BnST), and medial amygdala (MA). Lower levels were found in the ventromedial hypothalamic nucleus and cortical amygdala. The magnitude of the hybridization signal in the BnST and MPN was greater in males than in females. Treatment with T propionate significantly increased hybridization signal in BnST, MPN, and MA. These results confirm the anatomic distribution of P450arom mRNA within hypothalamic and limbic nuclei of the adult male rat and demonstrate that steady state concentrations are regionally regulated by T. Moreover, they demonstrate the necessity of using a molecular probe that can distinguish between P450arom variants in the brain.