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To develop a fast isolation method for arachidonic acid-producing fungi of high yield.Relation between the staining degree of mycelia of Mortierella alpina stained with triphenyltetrazolium chloride (TTC) and arachidonic acid content in the fungal lipids was investigated. Results showed that staining degree of mycelia stained with TTC increased when arachidonic acid content in mycelia lipids increased. This finding was used to isolate strains of high arachidonic acid yield. Arachidonic acid producing fungi were selectively isolated from soil at a low temperature of 4°C and the mycelia of these isolates were stained with TTC.The strain M. alpina M6 that had the highest staining degree had the highest arachidonic acid content (72·3%). The yield of arachidonic acid in this strain reached 4·82 g l−1.A fast and effective method to isolate strains of high arachidonic acid yield was established according to the finding that staining degree of mycelia of M. alpina was positively correlated with arachidonic acid content in mycelia lipid.