The heterotrophic marine alga Crypthecodinium cohnii is known to produce docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications, during batch cultivation on complex media containing sea salt, yeast extract, and glucose. In the present study, fed-batch cultivation was studied as an alternative fermentation strategy for DHA production. Glucose and acetic acid were compared as carbon sources. For both substrates, the feed rate was adapted to the maximum specific consumption rate of C. cohnii. In glucose-grown cultures, this was done by maintaining a significant glucose concentration (between 5 and 20 g/L) throughout fermentation. In acetic acid-grown cultures, the medium feed was automatically controlled via the culture pH. A feed consisting of acetic acid (50% w/w) resulted in a higher overall volumetric productivity of DHA (rDHA) than a feed consisting of 50% (w/v) glucose (38 and 14 mg/L/h, respectively). The rDHA was further increased to 48 mg/L/h using a feed consisting of pure acetic acid. The latter fermentation strategy resulted in final concentrations of 109 g/L dry biomass, 61 g/L lipid, and 19 g/L DHA. These are the highest biomass, lipid, and DHA concentrations reported to date for a heterotrophic alga. Vigorous mixing was required to sustain aerobic conditions during high-cell-density cultivation. This was complicated by culture viscosity, which resulted from the production of viscous extracellular polysaccharides. These may present a problem for large-scale industrial production of DHA. Addition of a commercial polysaccharide-hydrolase preparation could decrease the viscosity of the culture and the required stirring. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 666–672, 2003.