IN primary cultures of rat cerebral cortex, N-methyl-D-aspartate causes widespread neurotoxicity. Inhibitors of the nitric oxide generating the enzyme nitric oxide synthase has been shown to attenuate the effects of N-methyl-D-aspartate in a number of neuronal systems both in vivo and in vitro. In our experiments, the nitric oxide synthase inhibitor N-nitroarginine was ineffective at blocking neurotoxicity induced by N-methyl-D-aspartate. Cyclic guanine monophosphate, known to be synthesized in response to nitric oxide was demonstrably inhibited by identical treatments with N-nitroarginine in sister cultures. We conclude that although nitric oxide is produced in response to N-methyl-D-aspartate, it is neither necessary nor sufficient for neurotoxicity.