The effects of temperature, medium composition and concentration, and aeration on mycelial growth, lipid content and mycelial fatty-acid composition of Achlya flagellata were studied. At the temperatures tested (20, 25 and 30 C) there was no effect of temperature on mycelial growth other than a more rapid initiation of growth at 30 C. The yield of mycelium from the chemically defined medium was the same regardless of temperature. Similar results were obtained for lipid content. Varying the C: N ratio had no significant effect on either mycelial growth or lipid content. Mycelial growth was improved significantly by growth in peptone-yeast-extract-glucose (PYG) broth. Increasing the concentration of PYG medium resulted in an increase in mycelial growth but a decline in lipid content. Of the carbohydrates tested, glucose was the best for growth. Aerated cultures grew better but produced less lipid. In all experiments reduced mycelial growth was correlated with higher percent lipids. Aeration did result in a greater degree of unsaturation of fatty acids. Increase in temperature resulted in an increase in the degree of unsaturation. Palmitic, oleic, and arachidonic acids are the major fatty acids in the mycelial lipid extract.