The addition of hyaluronate to intact chick embryonic heart fibroblasts enriched with a hyaluronate-binding protein (HABP) stimulated phosphorylation of tyrosine and serine/threonine residues in cellular proteins. A protein complex containing a hyaluronate-binding protein (cell-HABP) was isolated from the cultured heart fibroblasts. The isolated complex (Mr approximately 1 x 10(6] contained phosphoproteins that exhibited protein kinase activity specifically stimulated by hyaluronate. Both tyrosine and serine residues in the protein complex were phosphorylated in response to this glycosaminoglycan. The hyaluronate-stimulated protein kinase activity was tightly associated with cell-HABP in vitro; enzyme activity co-immunoprecipitated with cell-HABP using a monospecific anti-HABP antibody and co-eluted with cell-HABP when chromatographed on a column of Sephacryl S-1000 in 2.0 M guanidine hydrochloride. The uniqueness of the cell-HABP-associated protein kinase activity was suggested by both its specific response to hyaluronate, relative to related glycosaminoglycans such as heparin and chondroitin sulfate or to growth factors such as epidermal growth factor or insulin, and its antigenic distinction from other protein kinases such as growth factor receptors. These results point to a new mechanism by which glycosaminoglycans, such as hyaluronate, may modify cell behavior.