Tumor necrosis factor alpha was found to rapidly phosphorylate the unique mammalian small heat shock protein hsp28 without impairing its cytoplasmic localization and without inducing the synthesis of the heat shock proteins. In contrast to the C-kinase-dependent phosphorylation of hsp28 in response to the tumor promoter phorbol-12-myristate-13-acetate, the heat- and tumor necrosis factor-mediated phosphorylation of this heat shock protein appears to occur independently of C kinase. These observations suggest that a C-kinase-independent phosphorylation of hsp28 may be an early event in the cellular action of tumor necrosis factor alpha.