We investigated the carbon acquisition of three marine microalgae, Skeletonema costatum, Phaeocystis globosa, and Emiliania huxleyi in response to different light regimes. Rates of photosynthetic O2 evolution and CO2 and HCO3- uptake were measured by membrane inlet mass spectrometry in cells acclimated to cycles of 16:8 light: dark (LD; h:h) and 12:12 LD and were compared with those obtained under continuous light. In addition, cellular leakage was estimated for different photoperiods and ambient CO2 concentrations during growth. Maximum rates of photosynthesis more or less doubled under LD cycles compared with continuous light. In S. costatum and E. huxleyi, a remarkably higher contribution of HCO3- to the overall carbon uptake was observed under LD cycles. In contrast, P. globosa did not change its CO2:HCO 3- uptake ratio in response to daylength. Half saturation concentrations (K1/2) for O2 evolution and inorganic carbon (Ci) uptake were also influenced by the photoperiod. Under LD cycles K1/2 values for photosynthesis in S. costatum and P. globosa were similar or higher compared with continuous light, whereas they were much lower in E. huxleyi. With the exception of CO2 uptake in E. huxleyi and P. globosa, affinities for Ci decreased under the LD cycles. Cellular leakage was highest for E. huxleyi and lowest for S. costatum and generally decreased with increasing CO2 concentration. Although this study confirms species-specific differences in the CO2-concentrating mechanisms (CCMs), the effect of daylength on CO2 and HCO3- uptake has hitherto not been described. We put forward the idea that variations in light condition influence the cellular carbon demand, thereby imposing a stronger control on CCM regulation than the naturally occurring changes in CO2 supply. © 2006, by the American Society of Limnology and Oceanography, Inc.