Three groups of weanling male rats were fed on a fat-free diet for 13 weeks. One group received only the fat-free diet (FF rats), the other 2 groups received the fat-free diet and a daily supplement of 2 energy% ethyl linoleate ([n-6] rats), or 2 energy% ethyl linolenate ([n-3] rats). Urinary excretion of prostaglandin E (PGE), immunoreactive arginine vasopressin (iA VP), and kallikrein were determined. PGE was quantitated with a radioimmunoassay having 4.9% cross-reactivity with prostaglandin E (PGE). After 4 weeks on the diet, water consumption and urinary iAVP excretion increased significantly in the FF rats and the (n-3) rats compared with the (n-6) rats. Urinary PGE excretion was the same for all 3 groups during the first 10 weeks; thereafter it decreased in FF rats and (n-3) rats compared with the (n-6) rats. There was no difference in urinary PGE excretion between the FF rats and the (n-3) rats, even though large differences were found in the percentage of arachidonic acid (20:4[n-6]), icosapentaenoic acid (20:5[n-3]), and icosatrienoic acid (20:3[n-9]) of total kidney fatty acids as well as of kidney phosphatidylinositol fatty acids. Fractionation of urine extracts on high performance liquid chromatography with radioimmunoassay detection indicated that (n-3) rats excreted very little PGE, if any. Urine output followed the same pattern, as did urinary PGE excretion. Urinary kallikrein was estimated at week 12 only. It was found to be significantly lower in FF rats and (n-3) rats. Increased water consumption and increased urinary iAVP excretion seem to be early symptoms (after 4 weeks) of EFA deficiency, whereas decreased urine output and decreased urinary PGE excretion occur much later (after 10 weeks). Two energy% linolenate supplementation to a fat-free diet did not change the appearance of any of the measured EFA-deficiency symptoms except for a slightly improved growth rate. There was no evidence of a significant urinary PGE excretion in spite of an extreme enrichment of kidney lipids with 20:5(n-3). It is suggested that urinary PGE is derived from precursors delivered from an arachidonic acid pool, which is rather resistant to restriction in dietary linoleate.