The in vivo and in vitro effect of epinephrine on the incorporation and desaturation of [1-14C]eicosa-8,11,14-trienoic acid was studied in isolated adrenocortical cells of rats. Control cells incubated at different substrate concentrations and for different periods of time were able to incorporate eicosatrienoic acid and to desaturate it to arachidonic acid. The ultrastructural study demonstrated that most of the cells belonged to the zona fasciculata and presented a good preservation. When the cells were isolated from rats killed 7 and 12 h after the administration of a single dose of epinephrine, a decrease in the incorporation and desaturation of 20:3n6 was observed. The effect on the desaturation was independent from the incorporation of the acid, and it was also observed in the microsomes of the decapsulated adrenal gland from rats treated with epinephrine. The fine structure of adrenocortical cells did not show changes after the treatment with epinephrine. The addition of the hormone to the incubation medium containing cells isolated from untreated rats produced no effect on arachidonic acid biosynthesis, indicating that the effect of epinephrine would be indirect, through either a metabolic or a hormone mediator.