In our previous study, we demonstrated that porcine enamel matrix derivative (EMD) induces p21(WAF1/cip1) within 8 hours and subsequently arrests the cell cycle of human oral epithelial cells in G1 phase. In contrast, EMD markedly stimulates the proliferation of gingival fibroblasts without inducing p21(WAF1/cip1). To investigate the mechanism of how EMD produces these differential effects, we have focused on the initial response of these two cell types to EMD. In epithelial cell cultures, EMD stimulated cytoskeletal actin polymerization within 30 min and promoted cell adhesion within 3 hours, but EMD had no substantial effects on fibroblastic cell adhesion in our experimental system. EMD failed to stimulate either intracellular Ca2+ mobilization or CAMP production in either cell type. In both epithelial and fibroblastic cells, EMD (25-100 mug/ml) rapidly produced dose-dependent phosphorylation of the mitogen-activated protein kinase (MAPK) family: extracellular signal response kinase (ERK), p38-MAPK (p38-K), and c-Jun-terminal kinase/stress-activated protein kinase (JNK). However, neither inhibitors of MEK (ERK kinase) nor p38-K could block EMD's anti-proliferative action on epithelial cells. On the other hand, EMD rapidly stimulated translocation of smad2 into the nucleus in both cell types. Spurred by this finding, we assayed for TGF-beta1, a ligand for one receptor associated with smad2 activation, and detected significant levels in EMD preparations. The sutra of these pharmacological findings indicates that EMD contains at least one bioactive factor, which is most probably TGF-beta1 (or TGF-beta -like substances). In conjunction with the similarities in the differential growth-modulating actions between EMD and what is known for TGF-beta, we suggest that TGF-beta might act as the principal growth regulating agent of oral fibroblastic and epithelial cell types in EMD despite being present in only low levels.