Glycosaminoglycan (GAG) chains of proteoglycans (PGs) play important roles in fibrosis through cell-matrix interactions and growth factor binding in the extracellular matrix. We investigated the expression and regulation of PG core protein (versican) and key enzymes (xylosyltransferase [XT]-I, beta 1,3-glucuronosyltransferase [GlcAT]-I, chondroitin-4-sulfotransferase [C4ST]) implicated in synthesis and sulfation of GAGs in bleomycin (BLM) and adenovirus-transforming growth factor (TGF)-beta 1-induced lung fibrosis in rats. We also studied the role of GlcAT-I or TGF-beta 1 and the signaling pathways regulating PG-GAG production in primary lung fibroblasts isolated from saline-or BLM-instilled rats. The mRNA for XT-I, GlcAT-I, C4ST, and versican was increased in the lung 14 days after BLM injury. In vitro studies indicate that fibrotic lung fibroblasts (FLFs) expressed more XT-I, C4ST, and chondroitin sulfate (CS)-GAGs than did normal lung fibroblasts at baseline. TGF-beta 1 enhanced the expression of XT-I, C4ST-I, and versican in normal lung fibroblasts, whereas SB203580 or SB431542, by targeting p38 mitogen-activated protein kinase or TGF-b type-1 receptor/activin receptor-like kinase 5, respectively, attenuated the response to both TGF-beta 1 and FLFs on PG-GAG expression. Neutralizing anti-TGF-beta 1 antibody abrogated FLF-conditioned medium-stimulated expression of XT-I, GlcAT-I, versican, and CS-GAG. Forced expression of TGF-beta 1 in vivo enhanced versican, XT-I, GlcAT-I, and C4ST-I expression and PG-GAG deposition in rat lungs. Finally, induced expression of GlcAT-I gene in rat lung fibroblasts increased GAG synthesis by these cells. Together, our results provide new insights into the basis for increased PG-GAG deposition in lung fibrosis; inhibition of TGF-beta 1-mediated or fibrosis-induced PGGAG production by activin receptor-like kinase 5/p38 inhibitors may contribute to antifibrotic activity.