Both time-dependent modulation of intracellular signaling molecules and sequential induction of transcriptional regulators are essential for the differentiation of preadipocytes into adipocytes. We have now shown that the activity, but not the abundance, of p42/p44 mitogen-activated protein kinase (MAPK) is down-regulated during adipocyte differentiation. This decrease in p42/p44 MAPK activity does not appear to be a direct effect of hormonal inducers of differentiation but rather represents a characteristic event of adipocyte differentiation that is achieved through a persistent change in intracellular signaling. Although the phosphorylation or abundance of MEK, an upstream kinase for p42/p44 MAPK, was not altered during differentiation, the abundance of MAPK phosphatase-1 (MKP-1), a negative regulator of p42/p44 MAPK, was increased with a time course similar to that of the down-regulation of p42/p44 MAPK activity. Ectopic expression of MKP-1 in preadipocytes reduced and depletion of endogenous MKP-1 in mature adipocytes increased the activity of p42/p44 MAPK. Prevention of the up-regulation of MKP-1 abundance in preadipocytes by expression of Mkp-1 antisense RNA resulted in persistence of p42/p44 MAPK activation and blocked differentiation, effects that were reversed by the MEK inhibitor PD98059. These results suggest that MKP-1 plays an essential role in adipocyte differentiation through down-regulation of p42/p44 MAPK activity.