Transforming growth factor-ß (TGF-ß) is a potent inducer of collagenase-3 (MMP-13) gene expression in human gingival fibroblasts, and this requires activation of the p38 mitogen-activated protein kinase pathway. Here, we have constructed recombinant adenoviruses harboring genes for hemagglutinin-tagged Smad2, Smad3, and Smad4 and used these in dissecting the role of Smads, the signaling mediators of TGF-ß, in regulation of endogenous MMP-13 gene expression in human gingival fibroblasts. Adenoviral expression of Smad3, but not Smad2, augmented the TGF-ß-elicited induction of MMP-13 expression. In addition, adenoviral gene delivery of dominant negative Smad3 blocked the TGF-ß-induced MMP-13 expression in gingival fibroblasts. Co-expression of Smad3 with constitutively active MKK3b and MKK6b, the upstream activators of p38, resulted in nuclear translocation of Smad3 in the absence of TGF-ß and in induction of MMP-13 expression. The induction of MMP-13 expression by Smad3 and constitutively active mutants of MKK3b or MKK6b was blocked by specific p38 inhibitor SB203580 and by the dominant negative form of p38a. These results show that TGF-ß-induced expression of human MMP-13 gene in gingival fibroblasts is dependent on the activation of two distinct signaling pathways (i.e. Smad3 and p38a). In addition, these findings provide evidence for a novel type of cross-talk between Smad and p38 mitogen-activated protein kinase signaling cascades, which involves activation of Smad3 by p38a.