Mesophyll cells, protoplasts, and protoplast extracts of Digitaria sanguinalis were used for comparative studies of light-dependent CO2 fixation. CO2 fixation was low without the addition of organic substrates. Pyruvate, oxaloacetate, and 3-phosphoglycerate induced relatively low rates (10 to 90 μmoles/mg chlorophyll·hr) of CO2 fixation when added separately. However, a highly synergistic relationship was found between pyruvate + oxaloacetate and pyruvate + 3-phosphoglycerate for inducing light-dependent CO2 fixation in the mesophyll preparations. Highest rates of CO2 fixation were obtained with protoplast extracts. Pyruvate, in combination with oxaloacetate or 3-phosphoglycerate induced light-dependent rates from 150 to 380 μmoles of CO2 fixed/mg chlorophyll·hr which are equivalent to or exceed reported rates of whole leaf photosynthesis in C4 species. Concentrations of various substrates required to give half-maximum velocities of CO2 fixation were determined, with the protoplast extracts generally saturating at the lowest substrate concentrations. Chloroplasts separated from protoplast extracts showed little capacity for CO2 fixation. The results suggest that CO2 fixation in C4 mesophyll cells is dependent on chloroplasts and extrachloroplastic phosphoenolpyruvate carboxylase.