The factors affecting the regulation of photosynthetic carbon fixation in diverse phytoplankton populations are not yet understood. To this end, we have measured the expression of the gene (rbcL) for the major carbon fixation enzyme, ribulose‐1,5‐bisphosphate carboxylase/oxygenase, in coastal phytoplankton populations off Cape Hatteras and in oligotrophic oceanic picoplankton near Bermuda. Using gene probes specific for the cyanobacterial/chloro‐phytic clade and the chromophytic clade (diatoms, chrysophytes, prymnesiophytes, and others) of Form I rbcL genes (“cyano” and “chromo” probes, respectively), we have measured rbcL messenger ribonucleic acid (mRNA) levels in size‐fractionated coastal waters, in a decktop diel incubator and a Lagrangian drifter study, and in vertical profiles in stratified, oligotrophic ocean water. In coastal waters influenced by estuarine plumes, an equal distribution of carbon fixation between the picoplankton and the micro/nannoplankton occurred, with cyano rbcL mRNA coinciding with Synechococcus counts in the <1‐μm fraction, with the majority of the chromo rbcL mRNA expression occurring in the larger sized phytoplankton fraction. In profiles of oligotrophic oceanic waters, the cyano rbcL mRNA was found in the upper water column (∼50‐m depth) and coincided with peaks in Synechococcus counts. The chromo rbcL mRNA was concentrated at the subsurface chlorophyll a (Chl a) maximum (∼85 m) and corresponded to red‐fluorescing cell counts, thought to be picoeucaryotes and diatoms. Photosynthetic carbon fixation and RUBISCO enzyme activity encompassed both cyano and chromo rbcL mRNA peaks, suggesting a near equal contribution to carbon fixation in the water column by these two phytoplankton clades. Both decktop diel incubator studies and a Lagrangian drifter study in coastal waters indicated cyano rbcL transcription in the morning and chromophytic rbcL transcription in the late afternoon/early evening. Thus, the two major clades of RUBISCO‐containing phytoplankton occupy separate niches in time, space, and cell size in the waters off Cape Hatteras. The factors determining such clade‐specific niches may include efficiency of nutrient utilization, differences in relative carboxylase/oxygenase activity (τ values) of cyano (Form IB) and chromophytic (Form ID) RUBISCOs, and differences in pigment composition/adaptation to light regimes. Additionally, we propose that chromo rbcL mRNA may be indicative of new production, whereas cyano rbcL mRNA correlates with recycled production in stratified, oligotrophic oceanic environments.