1. Vascular endothelial and smooth muscle cells generate nitric oxide (NO) via different nitric oxide synthase (NOS) isozymes. Activation of the endothelial constitutive NOS (ecNOS) contributes to the maintenance of cardiovascular homeostasis, whereas expression of the endotoxin- and cytokine-inducible pathway (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse which occurs during septic shock and antitumour therapy with cytokines. Since the cytoskeleton is involved in the activation of certain genes and in some effects of endotoxin in macrophages, we investigated the role of microtubules and microfilaments in the activation of the NO pathway in cultured vascular cells. 2. Depolymerization of microtubules by either nocodazole or colchicine prevented lipopolysaccharide (LPS)- and interleukin-1β-induction of NO-dependent cyclic GMP accumulation. Steady state levels of iNOS mRNA, assessed by Northern blot and RT-PCR, and iNOS protein, assessed by Western blotting, were also decreased by either colchicine or nocodazole treatment. 3. Taxol enhanced microtubule polymerization alone, and prevented microtubule depolymerization elicited by nocodazole and colchicine. Associated with its effect on microtubule assembly, taxol prevented the inhibitory effects of nocodazole and colchicine on cyclic GMP accumulation and iNOS mRNA levels. 4. Disruption of microfilaments by cytochalasins had no inhibitory effect on the activation of the inducible NO pathway. 5. In contrast to cytokine-stimulated smooth muscle cells, modulation of either microtubule or microfilament assembly did not affect the constitutive NO pathway in endothelial cells, as endothelial cell- and NO-dependent cyclic GMP accumulation in endothelial-smooth muscle co-cultures remained unchanged. 6. Our findings demonstrate that microtubules play a prominent role in the activation of the inducible NO pathway in response to inflammatory mediators in smooth muscle cells but not of the constitutive synthesis of NO in endothelial cells.