The effect of phthalate ester plasticizers on a variety of enzyme systems was studied in rats, rabbits and pigs. The feeding of di(2-ethylhexyl) phthalate (DEHP) to animals at levels from 0.1% to 1.0% in the diet resulted in diverse biochemical effects, such as inhibition of cholesterologenesis in liver, testes, and adrenal gland; inhibition of cholesterologenesis in brain and liver of fetal rats from DEHP-fed dams; decreased plasma cholesterol levels; decreased synthesis of hepatic phospholipid and triglyceride; increased fatty acid oxidation in isolated liver mitochondria; and a transient decrease in fatty acid oxidation in isolated heart mitochondria. The addition of DEHP to preparations of rat liver in vitro resulted in inhibition of cholesterologenesis, and its addition to isolated mitochondria from rat heart produced an inhibition of adenine nucleotide translocase. DEHP-feeding to rats and rabbits, however, did not affect platelet function as judged by collagen- and ADP-induced aggregation. The studies presented here indicate that the exposure of animals to phthalate esters can result in a significant perturbation of normal metabolism in liver, heart, testes, adrenal gland and brain and can affect blood lipid levels.