A new 4-deoxyphorbol triester (4-DPT) was isolated from the lipophilic phase of the latex sap of E. biglandulosa Desf. Its strong inhibitory activity on oxidative phosphorylation in intact isolated liver mitochondria and the NADH-oxidase system in electron transfer particles of beef heart was studied. The ADP-stimulated respiration of isolated mitochondria was progressively inhibited by increasing concentrations of 4-DPT (IC 50 for succinate: 2·5 × 10 −8 mole/mg protein and IC 50 for glutamate: 1·1 × 10 −7 mole/mg protein). This effect was protein-dependent. The state-4-respiration and coupling efficiency of oxidative phosphorylation remained unaffected. The NADH-oxidase system of electron transfer particles was inhibited (IC 50 = 9·4 × 10 −5 M), while the succinate-cytochrome c oxidoreductase activity remained unchanged. When 4-DPT was oxidized at its unsaturated ester residue, glutamate oxidation of intact mitochondria was no longer influenced while the NADH oxidase system of electron transfer particles was inhibited as strongly as by the natural 4-DPT. The adenine nucleotide translocation as well as the succinate transfer through the inner mitochondrial membrane were not affected by 4-DPT. The results demonstrate that two different mechanisms of action are involved: one concerns the inhibition of coupling side I of the respiratory chain, the other the utilization of succinate resulting from an inhibitory action of 4-DPT somewhere between the transfer of succinate through the inner mitochondrial membrane and its oxidation at coupling side II. Our findings may explain the poisonous in vivo effects of 4-DPT.