Previous work has demonstrated that the activity of the enzyme cholesterol sulfotransferase is rapidly and dramatically increased upon squamous differentiation of a variety of epithelial cells in culture, including epidermal keratinocytes. As a step toward understanding the molecular mechanisms underlying this differentiation-related change, we now report the partial purification and characterization of this enzyme activity from rat skin. Supernatant solutions from rat skin homogenates were subjected to a series of column chromatography steps including anion exchange, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The purification procedure resulted in cholesterol sulfotransferase activity purified 2700-fold with a 11% recovery. The most purified preparation yielded a major Coomassie blue-stained band on denaturing polyacrylamide gel electrophoresis of an apparent molecular weight (MW) of 40 000 Da. Photoaffinity labeling with the donor substrate, 3'-phosphoadenosine-5'-phospho-[35S]-sulfate resulted in a single radiolabeled protein band on denaturing polyacrylamide gel electrophoresis, again of apparent MW 40 000 Da, strongly suggesting that the major Coomassie blue-stained band in the most purified preparation is the cholesterol sulfotransferase protein. Among 3β-hydroxysteroids with a Δ5 double bond that were tested, each served as a substrate, while androgens, estrogens, corticosteroids, p-nitrophenol and DOPA did not serve as substrates. Apparent Michaelis constants for the 3β-hydroxysteroid substrates ranged from 0.6 to 8 µM.Key words: sulfotransferase, ichthyosis, cholesterol, skin, enzymology.