In response to hormonal stimulation quiescent 3T3-L1 preadipocyte cells reenter the cell cycle and undergo a mitotic expansion phase prior to terminal differentiation. The cell cycle regulatory proteins p130 and p107 undergo dramatic changes in protein levels within 24 h of differentiation. The role of these proteins in regulating adipocyte mitotic clonal expansion and/or differentiation are unclear. It has recently been demonstrated that adipocyte proliferation can be uncoupled from adipocyte differentiation through the use of the pharmacological MEK inhibitor PD98059 or the tyrosine phosphatase inhibitor, sodium vanadate. We examined the expression of p130 and p107 in stimulated 3T3-L1 cells in the presence of either PD98059, U0126 or sodium vanadate. While inhibition of MEK blocked proliferation, the cells underwent differentiation normally. In contrast, vanadate blocked differentiation without affecting proliferation. Inhibition of MEK did not affect the increase in p107 expression in stimulated cells indicating that induction of p107 is independent of MAP kinase signaling. Vanadate treatment caused a significant delay in p107 expression in the first 24 h following stimulation. Under these conditions, p130 expression was relatively unchanged. Our results indicate that a rapid increase in p107 expression correlates with a commitment to undergo adipocyte differentiation. The data further suggest that the rapid induction of p107 is not required for cellular proliferation during the mitotic clonal expansion phase. Taken together, these findings provide correlative data that implicate p107 in the terminal differentiation, but not proliferation, of quiescent preadipocytes following hormonal stimulation.