Until recently, biodiesel production has been derived from terrestrial plants such as soybean and canola, leading to competition between biodiesel production and agricultural production for source materials. Microalgae have the potential to synthesize 30 times more oil per hectare than terrestrial plants without competing for agricultural land. We examined four genera (Cyclotella, Aulacoseira, Fragilaria, Synedra) of common freshwater diatoms (Bacillariophyceae) for growth and lipid content in defined medium (sD11) that replicates hypereutrophic conditions in lakes and wastewater treatment plant effluents and optimized the medium for silicon content. Cyclotella and Aulacoseira produced the highest levels of total lipids, 60 and 43 μg total lipids/ml, respectively. Both diatoms are rich in fatty acids C14, C16, C16:1, C16:2,7,10, and C22:5n3. Of the diatoms examined, Cyclotella reached the highest population density (>2.5 × 10(6) cells/ml) in stationary phase when many of the cells appeared to be filled entirely with oil. Silicon enrichment studies indicated that for optimal utilization of phosphorus and nitrogen by diatoms growing in wastewater effluent, the amount of silicon present or added to the effluent should be 17.5 times the mass of phosphorus in the effluent. With high growth rates, high lipid contents, and rapid settling rates, Cyclotella and Aulacoseira are candidates for biodiesel production.