<jats:p>Abstract. The oxygen isotopic composition (δ18O) of calcium carbonate of planktonic calcifying organisms is a key tool for reconstructing both past seawater temperature and salinity. The calibration of paloeceanographic proxies relies in general on empirical relationships derived from field experiments on extant species. Laboratory experiments have more often than not revealed that variables other than the target parameter influence the proxy signal, which makes proxy calibration a challenging task. Understanding these secondary or "vital" effects is crucial for increasing proxy accuracy. We present data from laboratory experiments showing that oxygen isotope fractionation during calcification in the coccolithophore Calcidiscus leptoporus and the calcareous dinoflagellate Thoracosphaera heimii is dependent on carbonate chemistry of seawater in addition to its dependence on temperature. A similar result has previously been reported for planktonic foraminifera, supporting the idea that the [CO32−] effect on δ18O is universal for unicellular calcifying planktonic organisms. The slopes of the δ18O/[CO32−] relationships range between –0.0243‰ (μmol kg−1)−1 (calcareous dinoflagellate T. heimii) and the previously published –0.0022‰ (μmol kg−1)−1 (non-symbiotic planktonic foramifera Orbulina universa), while C. leptoporus has a slope of –0.0048 ‰ (μmol kg−1)−1. We present a simple conceptual model, based on the contribution of δ18O-enriched HCO3− to the CO32− pool in the calcifying vesicle, which can explain the [CO32−] effect on δ18O for the different unicellular calcifiers. This approach provides a new insight into biological fractionation in calcifying organisms. The large range in δ18O/[CO32−] slopes should possibly be explored as a means for paleoreconstruction of surface [CO32−], particularly through comparison of the response in ecologically similar planktonic organisms. </jats:p>