Rat liver phosphorylase kinase activity was increased up to 7-fold by low concentrations of heparin, but other glycosaminoglycans were relatively ineffective. A half-maximal effect was seen with 0.5 units of heparin/ml, corresponding to 150 nM. The stimulation was rapid, i.e. occurring within 15 s, and was prevented or reversed by addition of the heparin-binding protein antithrombin III. The action of heparin was also sharply reduced by prior activation of the enzyme by MgATP. In the absence of heparin, the time course of the phosphorylase kinase reaction was concave upward, whereas in the presence of heparin, a constant rate was seen. The stimulatory effect of heparin was inversely proportional to the concentration of phosphorylase b. The data suggest that heparin reversibly stimulates a low activity form of liver phosphorylase kinase to a degree similar to that found with activation by phosphorylation.