Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine with a proposed role in obesity-related insulin resistance. This could be mediated by increased lipolysis in adipose tissue resulting in elevated free fatty acid levels. The early intracellular signals entailed in TNF-alpha-mediated lipolysis are unknown but may involve members of the mitogen-activated protein kinase (MAPK) family. We investigated the possible contribution of MAPK in TNF-alpha-induced lipolysis in human preadipocytes. TNF-alpha activated the three mammalian MAPK, p44/42, JNK, and p38, in a distinct time- and concentration-dependent manner. TNF-alpha also induced a concentration-dependent stimulation of lipolysis with a more than 3-fold increase at the maximal dose. Lipolysis was completely inhibited by blockers specific for p44/42 (PD98059) and JNK (dimetylaminopurine) but was not affected by the p38 blocker SB203580. Use of receptor-specific TNF-alpha mutants showed that activation of MAPK is entirely mediated by the TNFR1 receptor. The results in human preadipocytes differed from those obtained in murine 3T3-L1 adipocytes in which all three MAPK were constitutively active. Thus, studies of intracellular signaling pathways obtained in different cellular contexts should be interpreted with caution. In conclusion, although TNF-alpha activates all three known MAPK in human preadipocytes, only p44/42 and JNK appear to be involved in the regulation of lipolysis.