The mechanisms by which growth factors are degraded and the role this process plays in the regulation of cell growth are not well understood. Insulin degradation is believed to be mediated by a specific metalloprotease, insulin-degrading enzyme (IDE). We have previously shown that IDE can also degrade transforming growth factor-alpha (TGF alpha), but not epidermal growth factor (EGF), in vitro. This selectivity was surprising, since TGF alpha and EGF are structurally similar and bind to the same receptor with comparable affinities. Using a spectrum of protease inhibitors, we have now analyzed the degradation of TGF alpha, EGF, and insulin by human hepatoma HepG2 cells. The results suggest that bacitracin-sensitive metalloproteases are involved in the degradation of TGF alpha and EGF as well as insulin, and that the degradation of TGF alpha, but not EGF, is mediated in part by IDE. Inhibiting the activity of these metalloproteases decreased growth factor depletion, suggesting that these enzymes play an important role in the control of extracellular growth factor levels. The existence of separate degradative pathways for EGF and TGF alpha may explain how the two factors exert differential effects in some systems, and degradation of TGF alpha by IDE would provide a possible mechanism for interaction between the insulin and TGF alpha/EGF signalling systems.