Changes in the volumes of sexually dimorphic brain nuclei are often used as a biomarker for developmental disruption by endocrine-active compounds (EACs). However, these gross, morphological analyses do not reliably predict disruption of cell phenotype or neuronal function. In the present experiments, we used a more comprehensive approach to assess whether postnatal exposure to the EACs genistein (GEN) or bisphenol-A (BIS) affected the development of two sexually dimorphic brain regions in male rats: the anteroventral periventricular nucleus of the hypothalamus (AVPV) and the sexually dimorphic nucleus of the preoptic area (SDN). In addition to nuclear volumes, we also measured the number of immunopositive calbindin neurons in the SDN and the activational patterns of gonadotropin-releasing hormone (GnRH) neurons, a neuronal population that is functionally linked to the AVPV. In rats, exposure of the neonatal male brain to endogenous estrogen, aromatized from testicular testosterone, is essential for the proper sexual differentiation of these endpoints. Thus, we hypothesized that exposure to BIS and GEN during this critical period could disrupt brain sexual differentiation. Animals were given four subcutaneous injections of sesame oil (control), 250 microg GEN, or 250 microg BIS at 12 h intervals over postnatal days (PND) 1 and 2, gonadectomized on PND 85, and treated sequentially with estrogen and progesterone to stimulate Fos expression in GnRH neurons, a marker for their activation. A cohort of age-matched ovariectomized (OVX) females that were given the same hormone treatment in adulthood served as a positive control group. SDN volume was unchanged by treatment, but the number of calbindin neurons in the SDN was significantly increased by both BIS and GEN. GEN, but not BIS, demasculinized male AVPV volume, but patterns of GnRH neuronal activation were not affected by either compound. These results suggest that acute exposure to EACs during a critical developmental period can independently alter nuclear volumes of sexually dimorphic nuclei and their phenotypic profiles in a region specific manner.