Free fatty acids (FFA) in cerebrospinal fluid (CSF) are well-recognized markers of brain damage in animal studies. Information is limited regarding human CSF in both normal and pathological conditions. Samples of CSF from 73 patients, who had undergone lumbar puncture for medically indicated reasons, came from a core laboratory upon completion of ordered tests. Using high performance liquid chromatography, mean FFA concentrations (μg/L ± SEM) were: arachidonic 26.14 ± 3.44; docosahexaenoic 60.74 ± 5.70; linoleic 105.07 ± 10.98; myristic 160.38 ± 16.17; oleic 127.91 ± 10.13; and palmitic 638.34 ± 37.27. No differences in FFA concentrations were seen with gender, race, age, and/or indication for lumbar puncture. This is the first study to document normal human CSF FFA concentrations in a large series. Further characterization of FFA in pathological conditions may provide markers for evaluating clinical treatments and assisting in prognostication of neurological disease.