Human keratinocytes (NCTC 2544) in culture were labeled with equal amounts of either 14C-arachidonic acid, 14C-dihomo-gamma-linolenic acid or 14C-eicosapentaenoic acid. At various time points, the incubations were stopped and the distribution of the 14C-fatty acids was analyzed. All these eicosanoid precursor fatty acids were effectively incorporated into the cellular lipids of the keratinocytes, and the major radiolabeled individual lipid fraction was phosphatidylethanolamine. The distributions of arachidonic acid and dihomo-gamma-linolenic acid within cellular lipids were rather the same. However, less eicosapentaenoic acid than either arachidonic acid or dihomo-gamma-linolenic acid was incorporated into the phospholipids and, correspondingly, more eicosapentaenoic acid was incorporated into the nonphosphorus lipids. In the phosphatidylinositol + phosphatidylserine fraction, there was significantly less eicosapentaenoic acid than either arachidonic acid or dihomo-gamma-linolenic acid. The present study suggests that these eicosanoid precursor fatty acids are effectively incorporated into the human keratinocytes and that the pattern of incorporation and distribution of eicosapentaenoic acid appears to differ slightly from that of either arachidonic acid or dihomo-gamma-linolenic acid.