Three species of marine phytoplankton, Rhodomonas sp., Isochrysis galbana Parke, and Phaeodactylum tricornutum Bohlin, were cultivated in semicontinuous cultures to test biochemical responses (fatty acids; FAs) to five nitrogen (N):phosphorus (P) supply ratios and four growth rates (dilution rates). The characteristic FA profile was observed for each algal species (representing particular algal class), which remained relatively stable across the entire ranges of N:P supply ratios and growth rates. For all species, significant direct effects of N:P supply ratios on FAs were found at lower growth rates. The highest saturated and monounsaturated fatty acid (SFA and MUFA) contents were observed under N deficiency at the lowest growth rate in all three species, while responses of polyunsaturated fatty acids (PUFAs) revealed no consistent pattern. Total FAs (and SFAs and MUFAs) in all species showed significant negative correlations with N cell quota (QN) under N deficiency, but PUFAs had species-specific correlations with QN. The results show that characteristic FA profiles of algal genus or species (representing particular algal classes) underlie fluctuations according to culture conditions. The significant correlation between FAs and QN under N deficiency suggests that elemental and biochemical limitation of phytoplankton should be considered mutually as determinants of food quality for zooplankton in marine ecosystems.