We have previously demonstrated that porcine enamel matrix derivative (EMD) contains TGF-beta1 (or a TGF-beta-like substance). and that EMD rapidly translocates smad2, which is an effector of the TGF-beta signaling pathway. into the nucleus and modulates the proliferation of both human gingival fibroblastic and oral epitheiial cells in a cell type-specific manner. To investigate the involvement of TGF-beta in the growth modulatory action of EMD, two approaches have been used in the present study: i) a neutralizing anti-TGF-beta antibody to block EMD action, and ii) authentic porcine TGF-beta1 to compare with EMD. Both in epithelial and fibroblastic cells, TGF-beta1 closely mimicked EMD in nuclear accumulation of smad2, phosphorylation of MAP kinase family members, and consequent cell type-specific growth modulation, Anti-TGF-beta antibody, at levels which completely blocked TGF-beta1-induced smad2 translocation. strongly blocked EMD-induced smad2 translocation. This antibody also blocked other actions of EMD in epithelial cells, i.e. p38-MAP kinase (p38-K) phosphorylation, p21(WAF1/cip1) expression. and inhibition of DNA synthesis. In support of our previous proposal, these data suggest that TGF-beta1 (or a TGF-beta-like substance), which is delivered as a principal bioactive factor in EMD, inhibits epithelial cell proliferation probably by a smad2- mediated, p21(WAF1/cip1)-dependent mechanism. However, the same neutralizing antibody failed to convincingly block EMD-induced fibroblastic proliferation, which suggests that EMD may contain additional unidentified mitogenic factor(s), which act in combination with TGF-beta to fully stimulate fibroblastic proliferation.