Bovine adrenal phenylethanolamine N-methyltransferase is activated by Pi. Addition of potassium phosphate to the enzyme assay mixture increased the enzyme activity 5- to 8-fold depending on the purity of the enzyme. Neuraminidase treatment of the enzyme decreased the activation by 1/2. The activity of the enzyme was also increased by other negatively charged ions, including chloride and sulfate. The activation was less than 3-fold, and pretreatment with neuraminidase did not alter the degree of activation. The high degree of activation was specific to bovine adrenal enzyme. Rat adrenal enzyme was activated less than 2-fold by phosphate and not affected at all by chloride and sulfate. Bovine adrenal phenylethanolamine N-methyltransferase is apparently a glycosylated protein, containing sialic acid moieties, and this carbohydrate moiety plays a role in the activation of this enzyme. The difference in characteristics between bovine and rat adrenal phenylethanolamine N-methyltransferase is evidently due to posttranslational modification of the enzyme.