Brain aromatase has been considered to be an important clue in elucidating the actions of androgen on brain sexual differentiation. Using highly specific anti-P450arom antiserum, the regional and subcellular distributions were immunohistochemically evaluated in the preoptic, strial, and amygdaloid regions of developing rat brains. Aromatase-immunoreactive (AROM-I) neurons were classified into three groups. The first, in which immunostaining occurs only during certain pre- or neonatal days (E16-P2), included the anterior medial preoptic nucleus, the periventricular preoptic nucleus, neurons associated with the strial part of the preoptic area, and the rostral portion of the medial preoptic nucleus. The second is a striking AROM-I cell group in the "medial preopticoamygdaloid neuronal arc," which extends from the medial preoptic nucleus to the principal nucleus of the bed nucleus of the stria terminalis and the posterodorsal part of the medial amygdaloid nucleus. The AROM-I neurons appeared by E16, reaching a peak in staining intensity between E18 and P2 and diminishing after the perinatal stage. After P14, a third group of AROM-I neurons emerged in the lateral septal nucleus, the oval nucleus of the bed nucleus of the stria terminalis, and the central amygdaloid nucleus. The second group was thought to be the major aromatization center in developing rat brains, while the center might partly shift to the third group of neurons after the late infantile stage. The distribution and developmental patterns were basically similar in males and females, suggesting that the neonatally prominent aromatase is not induced by male-specific androgen surges occurring around birth. On immunoelectron microscopy, subneuronal aromatase was predominantly localized on the nuclear membrane and endoplasmic reticulum, which appeared to be appropriate for the efficient conversion of androgen into estrogen just prior to binding to the nuclear receptors.