Decreased nitric oxide (NO) formation, resulting from inhibition of NO synthase (NOS), may be important in the pathogenesis of ethanol central nervous system teratogenesis. The objectives of this study were to determine the ontogeny of NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the developing guinea pig, and to test the hypothesis that direct exposure to ethanol inhibits NOS activity in these brain regions at selected developmental ages. NOS activity was quantitated by an optimized radiometric assay. The ontogeny study demonstrated that NOS activity in the hippocampus and frontal cortex was not fully developed prenatally, and apparently increased during postnatal life to attain adult level of activity at postnatal day > 60. In the cerebellum, NOS activity increased during prenatal life to an apparent maximum in the mature near-term fetus at gestational day 63 (term, about 68 days), and then apparently declined during postnatal life to attain adult level of activity. In vitro ethanol exposure (25-100 mM) did not affect NOS activity in the hippocampus, frontal cortex, or cerebellum at any developmental age studied. These data indicate that, although the ontogeny of NOS activity varies between brain regions, ethanol does not directly affect NOS activity in the developing guinea pig. The effects of acute and chronic in utero ethanol exposure on NOS activity in these brain regions are currently being investigated.