AbstractAn essential fatty acid (EFA) deficiency‐like profile of fatty acids has been observed in HF‐1 human skin fibro‐blasts cultured at clonal densities in MCDB 110 and 0.4% fetal bovine serum (FBS). The profile was characterized by an accumulation of 16∶1n−7, 18∶1n−9, 20∶3n−9 and 22∶3n−9, a reduction of n−6 fatty acids and a reduction in total polyunsaturated fatty acids (PUFA). The fatty acid composition of sphingomyelin (SPH), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) was determined and, except for SPH, each displayed an EFA deficiency‐like profile. The triene to tetraene ratio (20∶3n−9/20∶4n−6) ranged from 5.3 in PI to 0.9 in PE. In addition, the highest percentage of 20∶3n−9 was present in the PI and the highest percentage of 22∶3n−9, in PE. Other human fibroblasts (normal, transformed and at different population doubling number levels [PDL] were grown under the same conditions and were found to display triene to tetraene ratios (20∶3n−9/20∶4n−6) in total cellular lipids ranging from 0.7 to 4.5. The accumulation of 20∶3n−9 and 22∶3n−9 is due primarily to the existence of a basal nutrient medium (MCDB 110) that allows for the rapid clonal growth of human fibroblasts at reduced serum levels (0.4%). This culture procedure can be exploited to further elucidate various aspects of lipid metabolism in human fibroblasts.