We have examined the relative rates of uptake of several fatty acids into washed, human platelets by measuring incorporation into cellular phospholipids. In the presence of 15 μM fatty acid-free albumin and with radioactive fatty acid concentrations of 5-500 nM, esterification into phospholipid was linear with time and platelet concentration and saturable with respect to fatty acid concentration. Two distinct classes of uptake rate were observed. Arachidonate and 5,8,11,14,17-eicosapentaenoate exhibited high affinity, relatively rapid incorporation into platelet phospholipids at pH 6.5: apparent Michaelis constant (K(m) = 30 nM, apparent maximum velocity (V(max)) = 28 pmol/min per 109 platelets. Two other eicosanoid precursors, 5,8,11-eicosatrienoate and 8,11,14-eicosatrienoate, exhibited the same V(max), but K(m) of 85 and 60 nM, respectively. Under the same conditions, stearate, oleate, and linoleate were incorporated into phospholipids much less efficiently (V(max) ~ 8 pmol/109 cells per min, apparent K(m) ≥ 170 nM). Qualitatively similar results were found at pH 7.4. Uptake of radiolabled, rapid-uptake fatty acids was not diminished by the presence of excess, unlabeled, slow-uptake fatty acids. Thus, the specificity of this esterification system resembles that of the arachidonate-specific, long-chain acyl-CoA synthetase present in platelets. It may represent the expression in vivo of the synthetase, although the apparent affinity of the synthetase for fatty acid is much less. This esterification system probably represents the physiologic mechanisms for platelet arachidonate uptake, whereby arachidonate is collected from plasma, despite the fact that its concentration is considerably lower than that of other plasma fatty acids.