Cholera toxin covalently ADP-ribosylates the a subunit of Gs proteins. The modified Gsalpha activates adenylate cyclase and leads to a dramatic increase in intracellular cAMP. The effect of cholera toxin on the production of tumor necrosis factor (TNF-alpha), a critical mediator of toxicity for a number of bacterial and viral infections, has not been examined. Here we show that cholera toxin stimulated human monocytes to secrete TNF-alpha. The subunit A of cholera toxin alone also induced TNF-alpha production, suggesting that TNF-alpha production is mediated through ADP-ribosylation activity of the toxin. Inhibitors of ADP-ribosylation such as 3-aminobenzamide and niacinamide blocked TNF-alpha induction. However, cyclic AMP analogs and adenylate cyclase activator forskolin did not induce TNF-alpha production in monocytes, suggesting that TNF-alpha induction is independent of cAMP. Furthermore, cholera toxin-induced TNF-alpha production was suppressed by protein kinase C inhibitors H7 and sphingosine and by phospholipase C inhibitors U73122 and ET-18-OCH3, suggesting that PLC and PKC mediate TNF-alpha induction. Cholera toxin-mediated induction of TNF-alpha occurs at the transcription level as demonstrated by the time-dependent expression of TNF-alpha mRNA. These results raise the possibility that TNF-alpha may play an important role in cholera toxin-mediated toxicity and demonstrate that cholera toxin activates TNF-alpha production through PLC-dependent and cAMP-independent pathways. The probable mechanisms of signal transduction from cholera toxin to PLC in monocytes will be discussed.