The methyl ester of prostaglandin F_2α was hydrolyzed by undiluted human serum with a t1/2 of about 5 min, and the ethyl ester was hydrolyzed at one third of this rate. The 2′-propyl and 3′-pentyl esters were de-esterified at a rapid initial rate and at a slower second rate beginning after 10 min incubation. Alterations at the carbon-15 position of prostaglandin F_2α such as 15(S)-15-methyl or the 15-acetate or 15-hexanoate resulted in a reduction in the rate of hydrolysis of the primary esters at carbon-1.Species variation in serum esterase was very large, with rat serum showing activity more than 500 times that of human serum. Rates of hydrolysis in monkey serum were lower than that of human, and activity in the peripheral blood of the dog was extremely low. Plasma esterase activity in the mesenteric blood of the dog was several times higher than that found in the plasma from the peripheral circulation.