The effect of ethanol on nitric oxide synthase (NOS) activity was examined histochemically and biochemically in cultured cerebral cells of chick embryos. The cells were isolated from 13- to 14-day-old chick embryos to which 10% ethanol (ethanol group) or saline (control) had been injected on the 3rd day of embryogenesis. Expression of NADPH diaphorase in cultured cells was stained using nitro blue tetrazolium (NBT). The activity of NOS was observed by the following 2 assays: NADPH diaphorase activity was determined using the substrate NBT with the cofactor NADPH, and NOS activity was determined by measurement as radiochemical activity of the conversion of [3H]L-arginine to [3H]L-citrulline. The number of isolated cells and viability from one cerebrum of chick embryo were 3 approximately 4 x 10(6) and more than 97%, respectively. In the neuronal cells, moderately positive expression of NADPH diaphorase was first detected on about the 3rd day of culture in both the control and ethanol group. The NADPH diaphorase and NOS activities in the isolated cells were higher in the ethanol group than in the control group. The NADPH diaphorase and NOS activities were significantly higher in the ethanol group than in the control group on the 4th and 2nd day of culture, respectively. These findings suggest that NO-released by elevated NADPH diaphorase activity and NOS activity is responsible for the induction of neuronal cell disorders attributed to chronic ethanol damage.