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The cryptophyte Rhodomonas is an important feed item for live feed organisms in aquaculture and although large-scale cultivation of Rhodomonas in photobioreactors (PBRs) is feasible, the production needs to be optimized through further studies of specific factors. Through small-scale experiments, several factors relevant for an on-going large-scale production of Rhodomonas were studied and the results presented here provide a useful insight on factors that can help future large-scale production. The content of polyunsaturated fatty acids (PUFAs) and the temporal sedimentation was compared in five strains of Rhodomonas. Strain K-1487 (R. salina) was chosen as the most suitable for cultivation in PBRs due to a good biochemical content of PUFAs and low cell sedimentation. The f/2 growth medium used for cultivation was modified by excluding CoCl2 which did not affect either growth rate or cell content of the PUFAs, DHA, EPA, and ARA. Furthermore, the growth medium was modified by adding the nitrogen source as ammonium (NH4+), nitrate (NO3−), urea, or combinations of these, with NH4+ yielding a significantly higher growth rate of 1.30 ± 0.07 day−1. The seawater used for cultivation was exposed to three types of treatments which gave no significant difference in the growth rate: (1) filtration (0.2 μm) + autoclaving, (2) filtration (0.2 μm) + UV-radiation, and (3) filtration (0.2 μm). Finally, the results for growth rates of inocula at initial densities ranging from 2000 to 200,000 cells mL−1 showed that growth rate decreased with increasing density but a final density of 106 cells mL−1 was obtained fastest with the highest initial density. With the present findings, several barriers for effective cultivation of Rhodomonas are solved and future large-scale production has become a great step closer.