Aim: To setup a convenient method for the measurement of free arachidonic acid (AA) in neurons and to analyze the effects of heat treatment on the AA levels in neurons so as to give a reference to the study on heat injury in neural system. Methods: Primary cultured striatum neurons of rats were treated at a temperature of 43°C for 1 h. Cells of the heat treated group and control group were homogenized, and the fatty acids in cell lysate and cell membrane were extracted respectively. After heptadecanoic acid (C17:0) was added as an internal standard, the levels of fatty acid methanol esters were detected with Gas/Mass Chromatography. Results: The relative abundant fatty acid, including saturated (palmitic acid and stearic acid) and unsaturated (oleic acid, linoleic acid and arachidonic acid) were detectable, and did not change significantly during heat treatment, either in cell lysate or in cell membrane. There were no significant difference between saturated acid and unsaturated acid. While the level of AA in cytoplasm increased 100% from 0.53 ± 0.13 to 1.00 ± 0.56 after heat treatment (F = 5.71, P < 0.05), but the level of AA in cell membrane was undetectable, even after heat treatment. Conclusion: More amount of AA is released from neurons in striatum induced by heat treatment, and the increased AA may be crucial in the injury signal transduction.