Sera from numerous animal sources contain enzymes that degrade the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). The activity was present in rat, mouse, guinea pig, rabbit and goat, and absent in fetal bovine, bovine, porcine, horse, chicken and human sera. The enzyme shows a marked specificity for hydrolysis of the long-chain 12-O acyl group, which results in the formation of phorbol-13-monoacetate as the major product. Enzymatic catalysis displayed two pH optima (5.5 and 8.0) and was completely destroyed by heat inactivation (56 degrees C, 30 min) of the serum. N-Ethylmaleimide and p-chloromercuribenzoate were relatively weak inhibitors, whereas NaF (5.0 mM) produced an approximate 80% reduction of hydrolysis. Esterase-lipase substrates, such as cholesterol oleate, alpha-naphthylacetate, alpha-naphthylpalmitate, and monotetradecanoylglycerol, were not inhibitory when added to the reaction mixture. Porcine pancreatic lipase does not catalyze the removal of the acyl or the acetyl groups from TPA.