Neurosteroids are a class of steroids synthesized de novo in the brain, several of which are potent modulators of GABA(A) receptor function. In developing brain GABA(A) receptor, stimulation plays a trophic role. Cortical levels of the GABAergic neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) vary dramatically across development; during the second week of life, elevated levels of 3alpha,5alpha-THP are associated with decreased GABA(A) receptor function. To determine whether alteration of endogenous 3alpha,5alpha-THP levels during development alters GABAergic interneurons in prefrontal cortex (PFC) at maturity, rat pups were exposed to 3alpha,5alpha-THP (10 mg/kg) on postnatal day 1 (P1), P2, and P5. On P80, frontal cortex tissue was assayed for GABAergic cell localization (parvalbumin and calbindin immunoreactivity), agonist-dependent [(3)H] dizocilpine (MK-801) binding to NMDA receptors in cortical homogenates, muscimol-mediated (36)Cl(-) influx into synaptoneurosomes, and 3alpha,5alpha-THP levels. The localization of parvalbumin-labeled cells was markedly altered; the ratio of cell number in the deep layers (V-VI) versus superficial layers (I-III) of adult PFC increased twofold in animals exposed to 3alpha,5alpha-THP on P1 or P5. Relative microtubule-associated protein-2 and calbindin immunoreactivity were not altered by perinatal 3alpha,5alpha-THP administration. Agonist-dependent [(3)H]MK-801 binding was decreased in PFC but not parietal cortex homogenates, whereas muscimol-mediated (36)Cl(-) influx and 3alpha,5alpha-THP levels were unchanged in frontal cortex of adult males exposed to 3alpha,5alpha-THP on P5. These data are consistent with a change in the distribution of a subset of interneurons in response to neurosteroid exposure and suggest that GABAergic neurosteroids are critical for normal development of GABAergic systems in the PFC.