Effect of culture conditions on biomass, lipid, and arachidonic acid production was investigated in the oleaginous fungus Mortierella alpina CBS 528.72 under shake flask conditions. Several factors have been found to affect the biomass buildup and lipogenesis in this fungus, complicated by the fact that different strains demonstrate varying optimization conditions. Growth, lipid accumulation, and arachidonic acid production in the strain investigated were influenced by media, pH, temperature, carbon source, nitrogen source, etc. The results indicated that the most effective medium for growth and arachidonic acid production was glucose yeast extract medium. The optimum pH and temperature were found to be 6.5 and 28°C, respectively. On the same weight basis, glucose was the most efficient carbon source for biomass and lipid production in this fungal strain which yielded 6.8 g/L dry biomass and 40.2% (w/w) total lipid after 7 days of cultivation. Maximum arachidonic acid (ARA) production of 40.41% achieved in rhamnose-containing media was not concomitant with higher biomass and lipid yields. Efficacy of organic carbon sources, viz, yeast extract and peptone over inorganic sources like sodium nitrate, ammonium sulfate, ammonium chloride, etc, was established in the present study. M. alpina CBS 528.72 grown in peptone acquired the highest lipid content (42.0% (w/w)). However, the ARA content (28.74%) proved to be significantly less than that grown in yeast extract (35.28%). Furthermore, it was found that the biomass and ARA production declined drastically in a medium with vegetable oils as the sole carbon source but triggered the lipogenic pathway leading to higher accumulation of total lipids. Under the ideal conditions mentioned above, the maximum biomass, total lipid, and arachidonic acid production were 6.8 g/L, 41.6%, and 35.28% total fatty acid, respectively, in shake flask system.