Previous work has shown that vitamin K regulates brain sulfotransferase activity. We now show that activity is lost after treatment with acid phosphatase and is restored with ATP. Orthophosphate activates sulfotransferase to a small degree (26%) with a maximum at 3 mM. Vitamin K1 (or menadione) + orthophosphate stimulates activity 400%; vitamin K alone is not active. ATP activates maximally at 10 mM; ATP + vitamin K increases activation 20%. Protein kinase inhibitors do not affect ATP or orthophosphate + vitamin K mediated activation. Sulfotransferase is inhibited by pyridoxal phosphate indicating modification of a lysyl residue.