The estrogen sulfotransferase activity of high-speed supernatants of mouse placenta and uterus behaves on conventional and high-performance liquid chromatographic gel filtration as an enzyme species with a molecular weight of the order of 30 000. This is so whether the cytosols are freshly prepared or have been stored at -20 degrees C before chromatography. The presence of thiol groups or EDTA has no effect on the elution pattern. The partially purified enzyme is extremely unstable and is poorly recovered by (NH4)2SO4 fractionation. Some stabilization can be achieved in the presence of 0.1 microM estradiol. Chromatofocusing of cytosols results in the elution of one or two sulfotransferase peaks, depending upon experimental conditions such as the presence or absence of thiol groups. These peaks act upon both estrone and estradiol as substrates. Chromatofocusing by fast protein liquid chromatography (FPLC) in the absence of thiol groups results in the elution of one sulfotransferase peak whose activity can be detected only when thiol groups are present during enzyme assay.