An impurity, probably an anion, present in some batches of the buffer substances 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), 2-morpholinoethane sulfonic acid (Mes) and piperazine-1,4-bis(2-ethane sulfonic acid (Pipes), activates the soluble 5'-nucleotidase from rat kidney. The affinity of the enzyme for 5'-IMP and the Vmax were both increased by the unidentified activator. ATP and 2,3-diphosphoglycerate, known activators of the soluble 5'-nucleotidase, had no effect if the incubation media were buffered with batches containing high concentrations of the activating impurity. These results suggest that the impurity interacts with the soluble 5'-nucleotidase at the same site as ATP and 2,3-diphosphoglycerate, however with a much higher affinity than these two compounds. It is possible that the same impurity might interfere with other proteins for which ATP is a substrate or a ligand.