Closed cultures of marine phytoplankton were established under variable conditions of CO2concentration, temperature, growth rate (by light limitation), andpH (but with nearly identical [CO2aq]) in order to assess the relative influence of these variables on the extent of carbon isotope fractionation relative to dissolved inorganic carbon sources. Culture biomass was not allowed to increase beyond levels that would significantly affect the dissolved carbon system in the closed cultures. In experiments withSkeletonema costatumandEmiliania huxleyi, increasing CO2concentrations led to increased carbon isotope discrimination (resulting in organic matter progressively depleted in δ13C, i.e., a greater, more negative ϵp). ϵpvalues forE.huxleyiwere 8–10‰ less than forS. costatumunder identical conditions. For theS. costatumcultures, there was nearly a 20 ‰ range in [CO2aq]‐dependent ϵp. The effect was nonlinear with a leveling off at high [CO2aq]. Over apH range of 7.5–8.3 but at a constant [CO2aq] there was a variation in carbon isotope fractionation byS. costatumof about 9 ‰ with a minimum atpH 7.8–7.9. There was a temperature effect of ∼8‰ on fractionation even after equilibrium temperature dependency of δ13C of CO2aq was taken into account. No growth rate effect was found forS. costatumover a modest range of growth rates. Culture experiments used to determine the carbon isotope fractionation by phytoplankton species must be conducted under well‐defined conditions of temperature,pH, and CO2concentrations. Hindcasts of ancient atmosphericpCO2from measurements of δ13C of organic carbon in marine sediments will require careful calibration because of the variety of possible facto