Using [³⁵S] PAPS as the sulfate donor, we have detected a sulfotransferase from bovine heart which catalyzes the sulfation of tyro-sine-containing peptides. The enzyme displayed optimal activity at pH 5.75 and 35°C in a one-hour reaction. The addition of 10 mM Mn²⁺ or Co²⁺ to the reaction mixture increased the sulfotransferase activity by 3.4-and 3.5-fold, respectively. In contrast, the maximum increment stimu-lated by Mg²⁺ was only 1.75-fold at 15 mM concentration, and instead of exerting an enhancement effect, Ca²⁺ was found to be a potent inhibitor. The addition of 50 mM NaF to the reaction mixture resulted in an increase in sulfotransferase activity of 3.3-fold. The Km for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) was determined to be 2 μM at a constant 0.5 mM Boc-Glu-Asp-Tyr-Val. Among the 10 peptides tested as substrates, Boc-Glu-Asp-Tyr-Val and Boc-Asp-Asp-Tyr-Val provided the highest activities.