Inclusion of sodium nitroprusside (Na2[Fe(2+)-(CN)5NO]) into the culture medium is toxic to cultured rat cerebellar granule neurons. A possible underlying mechanism may be the inhibition of phosphoinositide (PI) response to excitatory amino acids (EAAs) because activation of glutamate receptors can be neuroprotective and neurotrophic in differentiating neurons. Sodium nitroprusside selectively inhibited the PI response to EAAs (NMDA > glutamate = quisqualate > kainate) without affecting that to carbachol or KCl. In contrast, S-nitroso-N-acetyl-penicillamine (SNAP), another nitric oxide (NO) donor, potentiated NMDA-induced PI hydrolysis. Hemoglobin reversed the effects of nitroprusside and SNAP. However, NO may not be involved because NO solution was without effect and N-acetylpenicillamine, a SNAP analogue that does not contain a NO moiety, also potentiated NMDA-induced PI hydrolysis in a hemoglobin-sensitive manner. Furthermore, the metabolites of NO (nitrate and nitrite), L-arginine, reduced glutathione, 8-bromo-cyclic guanosine 3':5'-cyclic monophosphate (8-Br-cGMP), and atrial natriuretic peptide, which accelerates the production of cGMP independent of NO, were ineffective as modulators. However, potassium ferrocyanide (K4[Fe2+(CN)6]), but not potassium ferricyanide (K3[Fe3+(CN)6]), inhibited NMDA-induced PI hydrolysis as effectively as nitroprusside, but this inhibition was not reversed by hemoglobin. Cyanide, a product from the disintegration of nitroprusside, potentiated rather than inhibited NMDA-induced PI hydrolysis. Taken together, these results suggest that the parent molecule itself, nitroprusside, contributes primarily in inhibiting EAA-induced PI hydrolysis. Inhibition of EAA-induced PI hydrolysis may in part mediate the mechanisms of nitroprusside toxicity in primary cultures of differentiating cerebellar granule neurons.