Abstract A mucopolysaccharide-sulfating system from chick embryo cartilage has been localized in the 105,000 x g supernatant fluid prepared from cartilage homogenates. Adenosine triphosphate was an absolute requirement for sulfate incorporation, but inhibited at high concentrations; NAD+ stimulated sulfate incorporation but nucleotidyl sugars had little effect. Sulfate incorporation did not appear to be taking place into newly synthesized polysaccharide but rather into endogenous acceptors. Protamine chloride treatment of the supernatant fluid greatly diminished sulfate incorporation, which could be fully restored by boiled dialyzed supernatant fluid. A diverse group of polysaccharides was found to be capable of accepting sulfate, indicating that this sulfotransferase system is not highly specific.