A selection programme to increase the cellular eicosapentaenoic acid (EPA) content has been carried out with the microalga Isochrysis galbana. The selection process involved two stages of single selection. EPA content continuously increased from 2.4% dry weight (d.w.) of the ’parent’ culture to an average value of 5.3% d.w. in the final stage. The proportion of total EPA variation attributable to the genetic variation (heritability in a broad sense) was 0.99 showing the importance of the genome in the determination of this fatty acid. The growth and fatty acid profile of an EPA-rich isolate grown as a chemostat in a cylindrical photobioreactor have been studied. A decrease in EPA content was observed (5.21% w/w to 2.8% w/w) at the lowest dilution rate D = 0.024 h(-1), up close to the maximum growth rate, D = 0.038 h(-1). At the same time, the biomass concentration also decreased from 1015 mg/litre to 202 mg/litre over the above-mentioned range of dilution rate (D). Nonetheless, the EPA productivity increases with D, with a maximum of 15.26 mg/litre/day at D = 0.0208 h(-1). Furthermore, steady-state dilution rates may be related to average internal light intensity. Reverse-phase, high-pressure liquid chromatography (HPLC) on octadecylsilyl semi-preparative columns was used to separate stearidonic acid (SA), EPA and docosohexaenoic acid (DHA) in polyunsaturated fatty acid concentrate obtained by the urea complexation method from a fatty acid solution previously obtained by direct saponification of biomass. Isolate SA, EPA and DHA fraction purity was 94.8, 96.0 and 94.9%, respectively, with yields of 100.0, 99.6 and 94.0%.