In chondrogenesis, members of the transforming growth factor-beta (TGF-beta) superfamily play critical roles by inducing gene expression of cartilage-specific molecules. By using a chondrogenic cell line, ATDC5, we investigated the TGF-beta-mediated signaling pathways involved in expression of the aggrecan gene (Agc). At confluency, TGF-beta induced Agc expression within 3 h, and cycloheximide blocked this induction, indicating that de novo protein synthesis is essential for this response. At this stage, TGF-beta induced rapid, transient phosphorylation of Smad2, extracellular signal-activated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). Inhibition of the Smad pathways by transfection with a dominant negative Smad4 construct significantly reduced TGF-beta-induced Agc expression, indicating that Smad signaling is essential for this response. Furthermore, an inhibitor of the ERK1/2 pathway, U0126, or inhibitors of the p38 MAPK pathway, SB203580 and SKF86002, repressed TGF-beta-induced Agc expression in a dose-dependent manner, indicating that ERK1/2 or p38 MAPK activation is also required for TGF-beta-induced Agc expression in confluent ATDC5 cells. In differentiated ATDC5 cells, persistently high basal levels of ERK1/2 and p38 MAPK phosphorylation correlated with elevated basal Agc expression, which was inhibited by incubation with inhibitors of these pathways. Whereas Smad2 was rapidly phosphorylated by TGF-beta and involved in the initial activation of Agc expression in confluent cells, Smad2 activation was not required for maintaining the high level of Agc expression. Taken together, these results suggest an important role for transcriptional cross-talk between Smad and MAPK pathways in expression of early chondrocytic phenotypes and identify important changes in the regulation of Agc expression following chondrocyte differentiation.