The incorporation of [³⁵S]sulfate into heart proteoglycans has been studied in normal and alloxan-diabetic rats by perfusion and in vivo administration of the isotope; in the latter situation, comparison was also made of radiolabeled sulfate utilization by several other tissues (kidney, liver, lung, muscle, testes and skin). The radiolabeled products were characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis and anion exchange chromatography, as well as by sizing of the glycosaminoglycan chains by gel filtration both before and after nitrous acid treatment. The most prominent band observed in the heart guanidine extract by electrophoresis had a molecular weight of 85,000 and minor components (M_r=360,000 and 170,000) were also detected; approximately 20% of the proteoglycan associated [³⁵S]sulfate was present in heparan sulfate chains. After perfusion the pattern, as well as the amount of radioactivity recovered from the diabetic heart, was similar to the normal heart. In contrast, after intraperitoneal injection of the [³⁵S]sulfate, a substantial reduction in incorporation was found not only in heart but in several other tissues studied, although no qualitative differences were noted in the macromolecules formed by the two groups of animals. Measurement of the serum sulfate concentration indicated that the level in the alloxan-diabetic rat (1.23 mmol/l) was significantly less (p<0.01) than that of the normal rat (1.67 mmol/l). Moreover, when serum specific activity curves were prepared between 10 min and 24 h after isotope injection, the decline in the diabetic curve was found to be much more rapid than that of the normal curve, with the area under the diabetic specific activity curve between 0 and 4 h being 0.61 that of normal (p<0.005). Since the amount of free [³⁵S]sulfate recovered in the tissues was also considerably lower in the diabetic animals, the specific activity differences of the precursor appear to account for the reduced incorporation of sulfate observed in vivo in contrast to perfusion, where pool sizes and specific activity can be controlled. Measurement of renal sulfate clearance indicated that it is enhanced in the alloxan-diabetic rats, suggesting that handling of sulfate in diabetes is similar to that previously described in this disease for inorganic phosphate.