1 In the present study we examined whether interleukin-lf (IL-Ifl) increases the activity of adenylyl cyclase in vascular smooth muscle cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-KB (NF-KB). In addition the interaction between cyclic AMP-and cyclic GMP-elevating agonists on the IL-iIf-stimulated expression of iNOS was examined.2 Exposure of vascular smooth muscle cells to IL-,IB stimulated the formation of cyclic AMP but not of cyclic GMP. The intracellular level of cyclic AMP reached a maximum within 1 h and then gradually declined over the next 5 h. This IL-l, (60 u ml-')-stimulated formation of cyclic AMP was modest (about 3 fold at 60 u ml-' for 1 h) compared to that evoked by isoprenaline (about 9 fold at 3 x 10-6 M for 2 min). 3 The IL-iI (60 u ml1 for 24 h)-stimulated accumulation of nitrite, which was taken as an index of NO production, was concentration-dependently increased by preferential inhibitors of cyclic AMP-dependent phosphodiesterases (rolipram and trequinsin). This effect was reproduced by a specific activator of the cyclic AMP-dependent protein kinase(s) A, Sp-8-CPT-cAMPS (10-4 M) but was prevented by a specific inhibitor of cyclic AMP-dependent protein kinase(s) A, Rp-8-CPT-cAMPS (10-4 M). These compounds alone [rolipram (10-6 M), trequinsin (3 x 10-6 M) and Sp-8-CPT-cAMPS (l0-4 M)] slightly but significantly increased the release of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4 Inducible NOS protein was expressed in IL-/IB (30 u ml-', 24 h)-stimulated smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which had been exposed to IL-,IB in combination with either rolipram (3 x 10-6 M) or Sp-8-CPTcAMPS (l0-4 M) but was reduced in those exposed to IL-,IB and Rp-8-CPT-cAMPS (l0-4 M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to either Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS elicited no such effect. 5 Exposure of smooth muscle cells to IL-lI (30 u ml-') for 30 min increased the level of NF-KB-DNA complexes in nuclear extracts as detected by electrophoretic mobility shift assay. Similar levels of NF-KB-DNA complexes were found in cells which had been exposed to IL-,IB in combination with either Sp