Marine diatoms are responsible for up to 20% of global CO(2) fixation. Their photosynthetic efficiency is enhanced by concentrating CO(2) around Rubisco, diminishing photorespiration, but the mechanism is yet to be resolved. Diatoms have been regarded as C(3) photosynthesizers, but recent metabolic labeling and genome sequencing data suggest that they perform C(4) photosynthesis. We studied the pathways of photosynthetic carbon assimilation in two diatoms by short-term metabolic (14)C labeling. In Thalassiosira weissflogii, both C3 (glycerate-P and triose-P) and C4 (mainly malate) compounds were major initial (2-5 s) products, whereas Thalassiosira pseudonana produced mainly C3 and C6 (hexose-P) compounds. The data provide evidence of C(3)-C(4) intermediate photosynthesis in T. weissflogii, but exclusively C(3) photosynthesis in T. pseudonana. The labeling patterns were the same for cells grown at near-ambient (380 microL L(-1)) and low (100 microL L(-1)) CO(2) concentrations. The lack of environmental modulation of carbon assimilatory pathways was supported in T. pseudonana by measurements of gene transcript and protein abundances of C(4)-metabolic enzymes (phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase) and Rubisco. This study suggests that the photosynthetic pathways of diatoms are diverse, and may involve combined CO(2)-concentrating mechanisms. Furthermore, it emphasizes the requirement for metabolic and functional genetic and enzymic analyses before accepting the presence of C(4)-metabolic enzymes as evidence for C(4) photosynthesis.