The mechanism and possible regulation of C4 acid decarboxylation in NAD-malic enzyme-type C4 plants was studied using isolated bundle sheath cells and mitochondria from Panicum miliaceum. Rates of C4 acid-dependent photosynthetic O2 evolution equalled those observed with saturating NaHCO3; the rates ranged from 3 to 5 mumol min-1 (mg chlorophyll)-1. C4 acid-dependent O2 evolution required the addition of aspartate and 2-oxoglutarate (as a source of oxaloacetate) and also malate and orthophosphate. C4 acid decarboxylation by both isolated cells and mitochondria, measured as pyruvate production, also required all four of these components. The scheme previously proposed to account for aspartate decarboxylation in NAD-malic enzyme-type C4 plants does not envisage a role for externally derived malate. However, the mandatory requirement for malate (with orthophosphate), together with the observation that C4 acid decarboxylation is blocked by an inhibitor of the mitochondrial dicarboxylate transporter, suggests that a net flux of malate from outside the mitochondria is required to sustain this process. Arsenate was found to substitute for orthophosphate favoring a role for orthophosphate in malate transport rather than a metabolic one. The results are discussed in terms of likely mitochondrial metabolite transport mechanisms and regulation of the C4 acid decarboxylation process.