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The role of iron and light in controlling photosynthate production and allocation in phytoplankton populations of the Atlantic sector of the Southern Ocean was investigated in April–May 1999. The 14C incorporation into five biochemical pools (glucan, amino acids, proteins, lipids and polysaccharides) was measured during iron/light perturbation experiments. The diurnal Chl a-specific rates of carbon incorporation into these pools did not change in response to iron addition, yet were decreased at 20 μmol photons m−2 s−1, an irradiance comparable with the one at 20–45 m in situ depth. This suggests that the low phytoplankton biomass encountered (0.1–0.6 μg Chl a L−1) was mainly caused by light limitation in the deep wind mixed layer (>40 m). Regional differences in Chl a-specific carbon incorporation rates were not found in spite of differences in phytoplankton species composition: at the Antarctic Polar Front, biomass was dominated by a diatom population of Fragilariopsis kerguelensis, whereas smaller cells, including chrysophytes, were relatively more abundant in the Antarctic Circumpolar Current beyond the influence of frontal systems. Because mixing was often in excess of 100 m in the latter region, diatom cells may have been unable to fulfil their characteristically high Fe demand at low average light conditions, and thus became co-limited by both resources. Using a model that describes the 14C incorporation, the consistency was shown between the dynamics in the glucan pool in the field experiments and in laboratory experiments with an Antarctic diatom, Chaetoceros brevis. The glucan respiration rate was almost twice as high during the dark phase as during the light phase, which is consistent with the role of glucan as a reserve supplying energy and carbon skeletons for continued protein synthesis during the night.