The carbon isotopic composition of marine phytoplankton varies significantly with growth conditions. Aqueous CO2 concentration [CO2] and algal growth rate (μ) have been suggested to be important factors determining isotope fractionation (εp). Here we examine εp of the coccolithophorid Emiliania huxleyi in relation to CO2 concentration and light conditions in dilute batch cultures. Cells were incubated at different irradiance cycles, photon flux densities (PFDs), and [CO2]. Isotope fractionation varied between 6.7 and 12.3‰ under 16:8 h light:dark cycle (L:D) and between 14.7 and 17.8‰ at continuous light. εp was largely independent of ambient [CO2], varying generally by less than 2‰ over a range of [CO2] from 5 to 34 μmol L-1. Instantaneous carbon-specific growth rates (μc) and PFDs, ranging from 15 to 150 μmol m-2 s-1, positively correlated with εp. This result is inconsistent with theoretical considerations and experimental results obtained under constant light conditions, suggesting an inverse relationship between εp and μ. In the present study the effect of PFDs on εp was stronger than that of μ and thus resulted in a positive relationship between μ and εp. In addition, the L:D cycle of 16:8 h resulted in significantly lower εp values compared to continuous light. Since the observed offset of about 8‰ could not be related to daylength-dependent changes in μc, this implies a direct influence of the irradiance cycle on εp. These findings are best explained by invoking active carbon uptake in E. huxleyi. If representative for the natural environment, these results complicate the interpretation of carbon isotope data in geochemical and paleoceanographic applications.