Interleukin 1 (IL-1)-mediated gene regulation is dependent on cell-matrix interactions. Both IL-1-activated pathways, nuclear factor κB (NF-κB) and the stress-activated protein kinase (SAPK), can be regulated by cell adhesion and changes in the cytoskeleton, suggesting that cell-matrix effects on IL-1 responses are initiated in part though effects on signal transduction. Here we show that IL-1-induced transient alterations in cell shape and in the cytoskeleton in fibronectin attached cells are correlated with effects on peak activity of NF-κB and SAPK. Cells on fibronectin showed a 1.5-2-fold enhancement in IL-1-induced NF-κB activity compared with levels in cells on poly(L-lysine) or bare tissue culture plates. The effect was increased with increasing concentrations of fibronectin and was most prominent at lower concentrations of IL-1. In contrast, fibronectin attachment caused an approx. 50% decrease in the IL-1 activation of SAPK, eliminating the peak activity after 15 min of stimulation with IL-1. IL-1-induced NF-κB activity showed a successive, substratum-independent increase during 4 h of attachment and spreading, whereas the inhibitory effect of fibronectin on the SAPK pathway was induced at the initial stages of attachment. Further, the addition of a peptide containing the motif RGD resulted in a 40% decrease in NF-κB activity in cells on fibronectin, largely accounted for by an effect on the p50/p65 heterodimer. Similarly, blocking of integrin aggregation by RGD-containing peptide resulted in a total abrogation of the fibronectin effect on IL-1-induced SAPK activity. The results demonstrate disparate effects of cell adhesion on the activation by IL-1 of the NF-κB and SAPK pathways. Thus fibronectin attachment causes an up-regulation of NF-κB activity in the presence of IL-1, whereas in contrast it results in a pronounced decrease in IL-1-induced SAPK activity.