Human platelets contain at least two forms of phenol sulfotransferase (PST), a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which micromolar concentrations of phenol can serve as substrate. At higher concentrations phenol is also a substrate for the TL form. Studies of the regulation and the possible clinical value of measurements of platelet PST have been hampered because there is no specific substrate for the TS form of the enzyme. The purposes of these experiments were to determine whether there might be a better substrate than phenol for use in measurement of the activity of the TS form of platelet PST, and to attempt to physically separate the two forms of the platelet enzyme. The results of substrate kinetic, thermal stability, and inhibitor studies performed with platelet homogenates were all compatible with the conclusion that p-nitrophenol and 6-OH-melatonin were substrates for both the TS and TL forms of platelet PST. Norepinephrine, epinephrine and 5-OH-tryptamine were substrates for only the TL form. The apparent Km constants of the two forms of PST for p-nitrophenol differed by 7,100-fold when measured in platelet homogenates. This difference was 200 times greater than that which has been reported for phenol. Therefore, p-nitrophenol is the preferred substrate for measurement of the TS PST activity if interference by the TL activity is to be avoided. This information made it possible to use p-nitrophenol as a substrate in experiments designed to separate the two forms of platelet PST.(ABSTRACT TRUNCATED AT 250 WORDS)