We have previously described a family of benzamide derivatives that showed antiinflammatory activity in vivo on carragenin-induced paw edema and experimental cerebral edema. Those compounds inhibited eicosanoids production from activated macrophages (M phi) without inhibiting cyclooxygenase. To further investigate their antiinflammatory activity and compare it to that of classical cyclooxygenase inhibitors, we analyzed their effect on the production of a major proinflammatory cytokine, tumor necrosis factor (TNF-alpha), by in vitro-activated peritoneal macrophages. We show that, in marked contrast with ibuprofen, flurbiprofen and indomethacin which all significantly enhanced TNF production, the two benzamide derivatives tested, JM34 and JM42, significantly inhibited TNF-alpha production by zymosan or lipopolysaccharide-activated M phi. Those compounds did not interfere with the calcium-dependent pathway because they did not affect TNF production of either mice peritoneal M phi or human T cell clones induced by the calcium ionophore A23187 alone. More likely, these benzamide derivatives acted mainly at the level of the protein kinase C (PKC) pathway because: 1) After treatment of M phi with PKC inhibitors which significantly inhibited TNF production, our compounds showed no additional inhibition. 2) Our compounds significantly inhibited TNF production of M phi stimulated with the phorbol ester phorbol di-butyrate alone or in combination with A23187. 3) After depletion of PKC by prolonged phorbol di-butyrate treatment of M phi, inhibition of TNF production by our compounds was markedly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)