The effects of phorbol ester (TPA) and other known stimulators such as tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide on induction of mRNA for manganese-superoxide dismutase (Mn-SOD) were investigated in various cell lines. TPA enhanced Mn-SOD mRNA expression in TNF-resistant cell lines including HeLa cells, in which the other reagents also induced expression of the gene, but did not affect TNF-sensitive cells, in which the other stimulators did not alter expression of the gene. HeLa cells which had been desensitized to TPA by pretreatment with TPA for 24 h expressed Mn-SOD mRNA at a slightly higher level than the cells without TPA treatment. TPA-pretreated cells stimulated with TNF, however, expressed Mn-SOD mRNA at about twice the level of TNF-stimulated, TPA-untreated cells. When protein synthesis was inhibited by cycloheximide during TPA pretreatment, TNF no more enhanced the Mn-SOD mRNA accumulation. These data suggest that at least two separate signal-transducing pathways are involved in expression of this gene. One is triggered by protein kinase C activation itself in the absence of new protein synthesis. The other can be activated by stimulation with TNF, interleukin-1, or lipopolysaccharide and in which a protein factor that can be induced by TPA treatment is involved.