The preference of phytoplankton for ammonium over nitrate has traditionally been explained by the greater metabolic cost of reducing oxidized forms of nitrogen. This “metabolic cost hypothesis” implies that there should be a growth disadvantage on nitrate compared to ammonium or other forms of reduced nitrogen such as urea, especially when light limits growth, but in a variety of phytoplankton taxa, this predicted difference has not been observed. Our experiments with three strains of marine Synechococcus (WH7803, WH7805, and WH8112) did not reveal consistently faster growth (cell division) on ammonium or urea as compared to nitrate. Urease and glutamine synthetase (GS) activities varied with nitrogen source in a manner consistent with regulation by cellular nitrogen status via NtcA (rather than by external availability of nitrogen) in all three strains and indicated that each strain experienced some degree of nitrogen insufficiency during growth on nitrate. At light intensities that strongly limited growth, the composition (carbon, nitrogen, and pigment quotas) of WH7805 cells using nitrate was indistinguishable from that of cells using ammonium, but at saturating light intensities, cellular carbon, nitrogen, and pigment quotas were significantly lower in cells using nitrate than ammonium. These and similar results from other phytoplankton taxa suggest that a limitation in some step of nitrate uptake or assimilation, rather than the extra cost of reducing nitrate per se, may be the cause of differences in growth and physiology between cells using nitrate and ammonium.