The aim of this study was to produce stearidonic acid (SDA, 18:4n-3) omega-3 concentrates from 25 % SDA soybean oil (SDASO) by enzymatic acidolysis. Substrates were prepared by chemical and enzymatic hydrolysis of SDASO. A 62 % SDA free fatty acid mixture (SDA-FFA) was obtained by low temperature crystallization of the chemical hydrolyzate while partial hydrolysis of SDASO by non-immobilized lipase AY 30 (Candida rugosa) yielded a 51 % SDA acylglycerol mixture (SDA-GLY). Reaction conditions for acidolysis between SDA-FFA and SDA-GLY were optimized using response surface methodology (RSM). Incorporation of SDA into SDA-GLY by immobilized Lipozyme RM IM (Rhizomucor miehei) and non-immobilized Lipomod 34P-LO34P (C. cylindracea [rugosa]) lipases were modeled under varying levels of the substrate molar ratio (S r ), temperature (T), and time (t). Optimal conditions for production of a 60 % SDA concentrate were predicted to be S r = 4.8, T = 65 °C and t = 8 h for Lipozyme RM IM and S r = 5.0, T = 43 °C and t = 48 h for Lipomod 34P-L034P. The model was verified experimentally by gram scale synthesis under optimal conditions which resulted in the production of 59.98 and 58.98 % SDA concentrates (≥96 % triacylglycerols) by Lipozyme RM IM and Lipomod 34P-L034P, respectively.