Marked changes in the structure and metabolism of lipids were observed at scaly lesions induced in the skin of rats by the feeding for four months of a diet deficient in essential fatty acids. The concentration of neutral lipids increased in the experimental skin specimens with the exception of free cholesterol. Analysis of fatty acids in the skin lipids of these essential fatty acid deficient rats showed a marked increase in monoenoic and eicosatrienoic acids, with a concomitant decrease in linoleic and arachidonic acids. Incubation of skin specimens from the essential fatty acid deficient rats with [2-3H]glycerol and [1-14C]acetate showed a marked increase in the incorporation of both precursors into all lipid classes. Particularly, their incorporations into phosphatidylcholine were predominant compared to other lipid classes. With regard to the molecular species of phosphatidylcholine, saturated-monoenoic and monoenoic-monoenoic species were highly synthesized in the skin from essential fatty acid deficient animals, and the synthesis of saturated-dienoic and tetraenoic species was very low with either precursor. Significant decreases in these labelings were also noted in saturated-saturated species. Addition of prostaglandin E2 to the incubation medium did not significantly affect the metabolism of any of the lipid classes in the skin from the essential fatty acid deficient rats. The present results, therefore, suggest that phosphatidylcholine containing linoleic acid may be a key lipid in the epidermal barrier function.