Growth of adipose tissue involves differentiation of preadipocytes into mature lipid-containing adipocytes. The matrix metalloproteinases (MMPs) are known regulators of adipose tissue biology, and previous studies suggested a key role for gelatinase B (MMP-9) in adipogenesis. In the present study we have evaluated a potential functional role of MMP-9 by performing gene silencing in 3T3-F442A preadipocytes. At the end of a 12-day differentiation period, no significant differences were observed between MMP-9 knockdown cells and the control cells with respect to intracytoplasmatic lipid content, or expression of the adipogenic markers aP2, PPARγ, Lpl, and adiponectin, or of the preadipocyte marker Pref-1. Thus, in vitro differentiation of 3T3-F442A preadipocytes into mature adipocytes is not significantly affected by the loss of MMP-9.