This study has examined the acyl specificity of incorporation of polyunsaturated fatty acids into cellular glycerolipids of human skin fibroblasts. At low exogenous fatty acid concentrations (0.2-1.2 microM) the extent of incorporation of arachidonate, eicosapentaenoate, 8,11,14-eicosatrienoate and 5,8,11-eicosatrienoate is 60-150% greater than that of oleate or linoleate. As the concentration of exogenous free fatty acid is increased to 25 microM, there is little decrease in the percentage of exogenous oleate incorporated into cellular glycerolipids. Under these conditions, the percentage incorporation of arachidonate and eicosapentaenoate drops 2-3-fold and approaches that of oleate. In contrast, the percentage incorporation of 8,11,14-eicosatrienoate remains high as exogenous fatty acid concentrations are increased. Incorporation of arachidonate, eicosapentaenoate, 8,11,14-eicosatrienoate and 5,8,11-eicosatrienoate is inhibited by addition of any of the other C20 polyunsaturated fatty acids but not by palmitate or oleate. C20 polyunsaturated fatty acids other than the eicosanoid precursors are also not effective inhibitors of arachidonate incorporation. The high affinity incorporation of C20 polyunsaturated fatty acids does not appear to be due to their selective esterification in any one class of cellular phospholipids. These results are compatible with a model of two pathways of fatty acids incorporation into mammalian cells. One pathway utilizes all exogenous long-chain fatty acids and, at least in fibroblasts, is not readily saturable. The second is a high-affinity, low-capacity uptake mechanism specific for arachidonate and other precursors of eicosanoids. The acyl specificity of this latter pathway appears to be similar to that of platelet arachidonyl- CoA synthetase. Results obtained with 8,11,14-eicosatrienoate would indicate, however, that at high concentrations, additional mechanisms influence the acyl specificity of fatty acid incorporation in these cells.