A high content of linolenic acid lcads to reduction of keeping quality and frying stability of cooking oil. The present study was conducted to obtain a further reduction of linolenic acid in soybean [Glycine max (L.) Merr.] oil by re-irradiation of M-5 and determine its genetic system. M-5 is a mutant with 4.5% Iinolenic acid content, derived from the cultivar Bay (8.0% Iinolenic acid). A seed lot from line M-5 was treated with X-ray irradiation and M₂ plants were obtained from randomly selected seeds of Ml plants. The M₂ plants were screened for reduced linolenic acid. One plant was found with 3.0% Iinolenic acid content and was named MS382. The M₃ and M₄ generations of this line proved that the character was fixed and significantly lower than the M-5 control. For inheritance studies, MS382 was reciprocally crossed with M-5(fan) and LOLL[fanfanx^a, a recombinant of M-5 (fan)× M24 (fanx^a)]. The F₂ segregation ratio and the segregation of F₃ seeds from F₂ plants of MS382×M-5 indicated that reduced linolenic acid in MS382 was conbindly controlled by fan (M-5) and an additional gene. To determine if this additional gene was similar with the fanxa gene in LOLL, F₂ seeds and F₃ seeds from each F₂ plant of MS382×LOLL were evaluated. No transgressive segregation for linolenic acid was found in this cross, indicating the genes for reduced linolenic acid content in MS382 and LOLL were identical. However, the mutant MS382 was developed by re-irradiation which indicates the practicability of this technique to develop new gene for further reduction of linolenic acid in soybean oil.