In the development of high yield of eicosapentaenoic acid (EPA) by the diatom Nitzschia laevis, substrate limitation and toxic metabolite inhibition were the two principal obstacles. In the present study, a perfusion culture was developed to eliminate substrate (glucose) limitation and by-product inhibition simultaneously. Using an exponential feed strategy and manipulating glucose supply rate and by-product removal rate, the optimal glucose concentration in the feed medium was determined to be 50 g 1(-1). With this feed glucose concentration (S-0 = 50 g 1(-1)), the perfusion culture resulted to a cell dry weight concentration of 40 g 1(-1). Such a high cell density led to an EPA yield of 1112 mg 1(-1), which is the highest ever reported in microalgal cultures. In addition, the perfusion culture (at S-0 = 50 g 1(-1)) greatly reduced the amount of wastewater (broth) during cultures. Considering the simple set-up and operation, the proposed perfusion culture is potentially useful for large scale EPA production by N. laevis. (C) 2002 Elsevier Science Ltd. All rights reserved.