The microalga Pavlova lutheri is a potential source of economically valuable docosahexaenoic and eicosapentaenoic acids. Specific chemical and physical culture conditions may enhance their biochemical synthesis. There are studies relating the effect of CO2 on growth; however, this parameter should not be assessed independently, as its effect strongly depends on the light intensity available. In this research, the combined effects of light intensity and CO2 content on growth and fatty acid profile in P. lutheri were ascertained, in order to optimize polyunsaturated fatty acid production. The influence of the operation mode was also tested via growing the cultures by batch and by continuous cultivation. Higher light intensities associated with lower dilution rates promoted increases in both cell population and weight per cell. Increased levels of CO2 favored the total lipid content, but decreased the amounts of polyunsaturated fatty acids. Mass productivities of eicosapentaenoic acid (3.61∈±∈0.04 mg • L-1 • d-1) and docosahexaenoic acid (1.29∈±∈0.01 mg • L-1 • d-1) were obtained in cultures supplied with 0.5% (v/v) CO2, at a dilution rate of 0.297 d-1 and a light intensity of 120 μE • m -2 • s-1.