Effects of various cultural conditions on biomass, lipid and Gamma-linolenic acid (GLA) production were investigated in the oleaginous fungus Cunninghamella blakesleeana-JSK2 isolated from soil. The GLA production was influenced by various factors such as growth condition (static and shaken), incubation time, pH, temperature, carbon and nitrogen sources. The results indicated that optimum GLA production (21 %) was obtained when the fungus was grown under shaken condition at 120 rpm for 6 days with optimum pH and temperature of 6 and 28°C ,respectively. Glucose and potassium nitrate enhanced the GLA production. Urea and sucrose were poor substances for biomass, lipid and GLA production.