The essential fatty acid, linoleic acid (LA), is required for the epidermal barrier and LA is also the precursor fatty acid for arachidonic acid (AA). Both fatty acids are imported from systemic sources, because AA is also not synthesized in the epidermis. The present studies were undertaken to compare the uptake and incorporation into cellular lipid of these fatty acids and to determine whether they compete with one another in these processes in relation to keratinocyte differentiation. The incorporation of [I4C]LA and/or [14C]AA into phospholipids and triglycerides was examined in keratinocytes cultured under submerged and lifted conditions. In submerged (less well-differentiated) cultures, more LA was incorporated into phospholipids than AA, while AA was incorporated into triglycerides to a greater extent. When given together, neither fatty acid influenced the total and/or relative uptake and lipid distribution of the other. Compared to submerged cultures, the uptake of LA increased 2-fold in lifted (differentiated) cultures, while the uptake of AA was unchanged. Lifting increased the proportion of AA incorporated into phospholipids, but did not alter the distribution of LA among phospholipids or triglycerides. These data suggest that the essential fatty acids, LA and AA, which are destined for different metabolic roles within the keratinocyte do not compete with one another during their uptake and distribution among cellular lipid species. Furthermore, as keratinocytes differentiate in culture, their increased requirement for LA for the synthesis of barrier lipids may be achieved through the preferentially enhanced uptake and lipid incorporation.