We examined the energetic dependency of the biochemical and physiological responses of Thalassiosira pseudonana Hasle and Heimdal. Chaetoceros gracilis Schütt, Dunaliella tertiolecta Butcher, and Gymnodinium sanguineum Hirasaka to NH4+, NO3−, and urea by growing them at subsaturating and saturating photon flux (PF). At subsaturating PF, when energy was limiting, NO3− and NH4+ grown cells had similar growth rates and C and X quotas. Therefore, NO3− grown cells used up to 48% more energy than NH4+ grown cells to assimilate carbon and nitrogen. Based on our measurements of pigments, chlorophyll-a-specific in vivo absorption cross-section, and fluorescence-chlorophyll a−1, we suggest that NO3−, grown cells do not compensate for the greater energy requirements of NO3− reduction by trapping more light energy. At saturating PF, when energy is not limiting, the utilization of NO3−, compared to NH4+ resulted in lower growth rates and N quotas in Thalassiosira pseudonana and lower N quotas in Chaetoceros gracilis, suggesting enzymatic rather than energetic limitations to growth. The utilization of urea compared to Nh4+ resulted in lower growth rates in Chaetoceros gracilis and Gymnodinium sanguineum (saturating PF) and in lower N quotas in all species tested at both subsaturating and saturating PF. The high C:N ratios observed in all urea-grown species suggest that nitrogen assimilation may be limited by urea uptake or deamination and that symptoms of N limitation in microalgae may be induced by the nature of the N source in addition to the N supply rate. Our results provide new eridence that the maximum growth rates of microalgae may be limited by enzymatic processes associated with the assimilation of NO3−, or urea.