Phenol sulfotransferases (PSTs) catalyze the sulfate conjugation of catecholamines and a variety of phenolic compounds. Thermolabile and thermostable forms of PST exist in human tissue. Blood component thermostable PST activities have proved useful as measures of the enzyme activities in other tissues such as the liver. The most thoroughly studied blood component is the human platelet, which contains both thermolabile and thermostable PST activities. Partial localization of PST activity in blood has been characterized only for thermolabile PST. We performed the studies reported here to define the cellular and subcellular localization of both thermolabile and thermostable PST activities in blood elements. Blood samples from four adults were pooled and aliquots for platelet studies were anticoagulated with ethylenediaminetetraacetic acid. Aliquots for studies of granulocytes, mononuclear cells, and erythrocytes were defibrinated to avoid platelet contamination and were separated through Ficoll-Hypaque gradients. Cytosol thermolabile PST activities assayed with dopamine as the substrate and expressed as a percent of the total thermolabile PST activity per milliliter of whole blood were as follows: platelets, 97%; granulocytes, 0.6%; mononuclear cells, 0.7%; and erythrocytes, 0.4%. Cytosol thermostable PST activities measured with p-nitrophenol were as follows: platelets, 77% of the total activity; granulocytes, 19%; mononuclear cells, 1.2%; and erythrocytes, 0.5%. Plasma and membrane-bound activities were less than 2.3% of total activities for each form. Because granulocyte thermostable PST was present in an amount greater than expected, it was further characterized. The Michaelis-Menten constant values for p-nitrophenol and 3'-phosphoadenosine-5'-phosphosulfate were 1.13 mumol/L and 0.6 mumol/L, respectively. The pH optimum of 6.6, a 50% inhibitory concentration for 2,6-dichloro-4-nitrophenol of 1.0 mumol/L, and retention of 56% of activity after preincubation at 45 degrees C for 15 minutes were the same for the granulocytes as for platelet thermostable PST. In summary, our study confirms and extends our knowledge of localization of blood thermolabile PST. Our data define for the first time the localization of blood thermostable PST and highlight the substantial contribution of granulocyte thermostable PST activity. Granulocytes represent an easily obtained nucleated cell for the study of human thermostable PST.