Superinduction of cyclooxygenase-2, in murine RAW 264.7 macrophages as well as human pulmonary type II A549 epithelial cells, is achieved by the simultaneous addition of agonists such as lipopolysaccharide or interleukin-1beta and the NO(*) donor S-nitrosoglutathione. NO(*)-evoked superinduction of cyclooxygenase-2 in the presence of agonists was dose-dependent and required transcriptional as well as translational regulation. We sought to further analyze NO(*)-elicited superinduction at the level of the transcription factor NF-kappaB that is obligatory for cyclooxygenase-2 expression. NO(*)-mediated NF-kappaB activation was restricted to low concentrations of S-nitrosoglutathione (50-200 microM), while a higher dose of S-nitrosoglutathione (1 mM) was ineffective. Not observing a correlation between NF-kappaB activation and cyclooxygenase-2 expression under NO(*)-delivery stimulated our interest in analyzing AP-1. NO(*) efficiently activated AP-1 at all concentrations tested. The involvement of AP-1 in promoting cyclooxygenase-2 superinduction was established in cells transfected with the dominant-negative c-Jun mutant, TAM-67. Enhanced expression of cyclooxygenase-2 by lipopolysaccharide/S-nitrosoglutathione-treatment was attenuated in TAM-67 transfectants, while the response to lipopolysaccharide alone remained unaffected. We conclude that AP-1 activation exclusively conveys the NO(*) signal that is required for superinduction of cyclooxygenase-2. Superinduction of cyclooxygenase-2 is restricted to a situation where both, NF-kappaB and AP-1 are activated. Under inflammatory conditions this might be achieved by the costimulatory signals provided by agonist challenge and NO(*).