The effect of insulin on the oxidative desaturation of 1-14C palmitic acid to palmitoleic acid (delta 9 desaturase) 1-14C linoleic acid to alpha-linolenic acid (delta 6 desaturase) on rat liver microsomes and 1-14C eicosa-8,11,14-trienoic acid to arachidonic acid (delta 5 desaturase) on rat liver microsomes and hepatoma tissue culture (HTC) cells was studied. After 12 h of insulin injection, at a dose of 2.5-12.5 U/kg no change was found in delta 9 desaturation activity while delta 5 desaturation activity decreased. The conversion of linoleic to alpha-linolenic acid decreased when the amount of insulin injected was 5 U/kg or more. The effect of insulin (5 U/kg) on delta 9, delta 6 or delta 5 desaturation activity was tested from 1 to 12 h after the injection. The conversion of palmitic acid to palmitoleic acid showed no important changes along the time, while delta 5 desaturation activity decreased at all the times tested. delta 6 Desaturation activity showed a slight increase after 1 h of insulin treatment and then decreased significantly up to the end of the experiment. The addition of 400 mU/ml or more of insulin to the incubation medium of HTC cells produced a significant decrease on the conversion of eicosatrienoic acid to arachidonic acid. The effect of insulin on fatty acid desaturation activity of liver microsomes of normal rats differs from that of diabetic rats. The role of this hormone in relation to other hormones, carbohydrate metabolism and lipid biosynthesis on the activity of the desaturases was discussed.