Bovine epiphyseal plate chondrocytes were cultured by a method combining both suspension culture and high density monolayers. The matrix synthesized by the cultured cells was analyzed at fifteen days for glycosaminoglycan, proteoglycan, and collagen content. In the cell culture product glycosaminoglycan distribution was: 65% chondroitin-6-sulfate, 18% chondroitin-4-sulfate, 15% keratan sulfate, and less than 2% dermatan sulfate. Essentially all the radioactive sulfate in labelled specimens was present in high molecular weight aggregates. The collagen which was synthesized co-migrated with Type II collagen standard. Parallel analysis showed the matrix of cultured cells to be similar to that of intact epiphyseal plate tissue. This study demonstrates the ability to grow epiphyseal plate chondrocytes in a cell culture system which allows matrix synthesis similar to that seen in vivo.