Abstract: The formation of the sulfate donor [35S]3′-phos-phoadenosine 5′-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to β-naphthol under the action of phenolsulfo-transferase activity from rat brain cytosol, with the [35S]β-naphthyl sulfate formed being isolated by polystyrene bead c'iromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chroma-tography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of ∼7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1–5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micro-r olar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 188.8.131.52), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5′-phos-phosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity. Among subcellular fractions from cerebral cortex, [35S]PAPS formation only occurred in the cytosolic fractions, including that derived from hypoosmotically shocked synaptosomes. [35S]PAPS formation was not detectable in the crude cortical homogenate, and this was attributable to a thermolabile inhibitory effect of cerebral membranes toward the cytosolic synthesizing system.