Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has been implicated as a causative factor in obesity-linked insulin resistance. It is commonly accepted that macrophage-derived TNF-alpha acts in a paracrine manner on adjacent adipocytes to inhibit the expression of various adipocyte genes and to attenuate insulin signaling. Several studies have revealed that signal transducer and activator of transcription (STAT)5 proteins are modulated during adipogenesis and can modulate the transcription of some adipocyte genes. In this study, we demonstrate that TNF-alpha treatment, in the presence of cycloheximide, also results in the rapid turnover of STAT5A and STAT5B in a process that is independent of STAT5 activation by tyrosine phosphorylation. In addition, STAT5B is more labile than STAT5A under these conditions, suggesting that the COOH terminus of STAT5 may be involved in the turnover of each protein. Initial characterization of the TNF-alpha and cycloheximide-mediated degradation of STAT5 indicates that inhibition of the proteasome stabilizes both forms of STAT5 in the presence of TNF-alpha. In addition, the use of an NF-kappaB inhibitor results in the stabilization of STAT5A in the presence of TNF-alpha and cycloheximide, indicating that the degradation of STAT5 proteins under these conditions may involve the NF-kappaB pathway. STAT5 proteins are abundantly expressed in mature adipocytes and are normally extremely stable proteins under a wide range of conditions. However, our results demonstrate that the potentiation of TNF-alpha-mediated signaling in the presence of cyclohexmide is associated with a significant increase in the degradation of STAT5 proteins in 3T3-L1 adipocytes.