A series of polyunsaturated fatty acids (PUFAs), including 5,8,11,14,17-cis-eicosapentaenoic acid (EPA) and 5,8,11,l4-cis-arachidonic acid (ARA), have widespread nutritional and pharmaceutical value. This study investigated the potential production of the two economically important fatty acids with a fungal fermentation process. The substrates for the fungal fermentation process were crude soybean oil (SBO), a sucrose waste stream (SWS) and a soymeal waste stream (SMW). Glucose (GLU) was used as a substrate in control groups. The microorganisms used were Mortierella elongata NRRL 5513 and Pythium irregulare ATCC 10951. The use of P. irregulare ATCC 10951 is preferred since it produced high levels and reasonable ratios of EPA and ARA at various temperatures (12, 18 and 24 degrees C). An advantage of P. irregulare was its ability to produce EPA at room temperature, which is desirable for commercial applications. Soybean oil had a unique characteristic of stabilizing pH; the optimal initial pH was 6.0. An emulsifier, Tween 80, allowed much greater dispersion of the SBO in aqueous broth and helped increase EPA and ARA production. In experiments exploring the combination effects of sugars (1, 2 and 3%) with soybean oil (4%) and Tween 80 (0.2%) at 12, 18 and 24 degrees C, EPA yields of SMW+SBO were significantly higher than those of GLU+SBO and SWS+SBO. The greatest EPA production (1400 mg/l) was obtained at 12 degrees C (1% SMW, 4% SBO). Cultivation of II irregulare at reduced temperatures increased lipid unsaturation. The highest ARA level appeared at 18 degrees C-SMW+SBO (2000 mg/l), which was a statistically interactive temperature-media combination. The ARA/EPA ratio in this study ranged from 0.2 to 4.0, which would be reasonable for food additive or supplement applications, e.g infant formula.