Bovine heart valves metabolize up to 96% of exogenous <sup>14</sup>C-glucose to lactate when incubated in vitro under appropriate conditions. Small proportions of the <sup>14</sup>C-radioactivity are incorporated into glycogen, glycosaminoglycans and lipids and are oxydized to <sup>14</sup>CO<sub>2</sub>. Difference between the valves of the left ventricle (aortic and mitral valve) and of the right ventricle (pulmonary and tricuspid valve) were found in the rate of glycolysis as well as in the collagen, glycogen and glycosaminoglycan content. The in vitro incorporation of [U-<sup>14</sup>C] glucose radioactivity into the total glycosaminoglycans resulted in a specific labelling of hyaluronic acid, dermatan sulfate and chondroitin sulfate, but the specific radioactivities of the individual glycosaminoglycans and their relative rates of biosynthesis differed markedly. The relative rates of biosynthesis were 2.9–7.6 for hyaluronate, 1.0 for dermatan sulfate and 0.26–0.59 for chondroitin sulfate. After labelling the sulfated glycosaminoglycans with <sup>35</sup>S-sulfate, the metabolic heterogeneity of dermatan sulfate and chondroitin sulfate was confirmed, but, in addition, heparan sulfate – not previously detected in heart valves – was identified as a third sulfated glycosaminoglycan. Chemical analyses of heparan sulfate revealed a sulfate/uronic ratio of 0.5; its relative amount was found to be 2–4% of the total glycosaminoglycans. Analogous chemical and metabolic data were obtained with chordae tendineae, except that a higher relative percentage of dermatan sulfate was found than in the corresponding heart valves.