Microalgae offer a high potential for energetic lipid storage as well as high growth rates. They are therefore considered promising candidates for biofuel production, with the selection of high lipid-producing strains a major objective in projects on the development of this technology. We developed a mutation-selection method aimed at increasing microalgae neutral lipid productivity. A two step method, based on UVc irradiation followed by flow cytometry selection, was applied to a set of strains that had an initial high lipid content and improvement was assessed by means of Nile-red fluorescence measurements. The method was first tested on Isochrysis affinis galbana (T-Iso). Following a first round of mutation-selection, the total fatty acid content had not increased significantly, being 262 +/- 21 mgTFA (gC)(-1) for the wild type (WT) and 269 +/- 49 mgTFA (gC)(-1) for the selected population (S1M1). Conversely, fatty acid distribution among the lipid classes was affected by the process, resulting in a 20% increase for the fatty acids in the neutral lipids and a 40% decrease in the phospholipids. After a second mutation-selection step (S2M2), the total fatty acid content reached 409 +/- 64 mgTFA (gC)(-1) with a fatty acid distribution similar to the S1M1 population. Growth rate remained unaffected by the process, resulting in a 80% increase for neutral lipid productivity. Biotechnol. Bioeng. 2012; 109: 2737-2745. (c) 2012 Wiley Periodicals, Inc.