The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn‐over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF‐β), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP‐3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF‐β‐mediated induction of TIMP‐3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12‐myristate 13‐acetate (PMA), a PKC activator, increased TIMP‐3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP‐3 gene to TGF‐β. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF‐β induction of TIMP‐3. H7 and genistein also suppressed TGF‐β‐induced TIMP‐3 protein expression. These results suggest that TGF‐β signaling for TIMP‐3 gene induction involves H7‐sensitive serine/threonine kinase as well as herbimycin A‐ and genistein‐sensitive protein tyrosine kinases. J. Cell. Biochem. 70:517–527, 1998. © 1998 Wiley‐Liss, Inc.