The glia-derived, neurotrophic protein S100 beta has been implicated in development and maintenance of the nervous system. However, S100 beta has also been postulated to play a role in mechanisms of neuropathology, because of its specific localization and selective overexpression in Alzheimer's disease. To begin to address the question of whether S100 beta can induce potentially toxic signaling pathways, we examined the effects of the protein on nitric oxide synthase (NOS) activity in cultures of rat cortical astrocytes. S100 beta treatment of astrocytes induced a time- and dose-dependent increase in accumulation of the NO metabolite, nitrite, in the conditioned medium. The S100 beta- stimulated nitrite production was blocked by cycloheximide and by the NOS inhibitor N-nitro-L-arginine methylester, but not by the inactive D-isomer of the inhibitor. Direct measurement of NOS enzymatic activity in cell extracts and analysis of NOS mRNA levels showed that the NOS activated by S100 beta addition is the calcium-independent, inducible isoform. Furthermore, the specificity of the effects of S100 beta on activation of NOS was demonstrated by the inability of S100 alpha and calmodulin to induce an increase in nitrite levels. Our data indicate that S100 beta can induce a potent activation of inducible NOS in astrocytes, an observation that might have relevance to the role of S100 beta in neuropathology.