There are three major sub-types of C4 plants based on the dominant decarboxylating enzymes in their bundle sheath cells (BSC). Three different enzymes are involved in releasing CO₂ in BSC: NAD-malic enzyme (NAD-ME), phosphoenol pyruvate carboxykinase (PEP-CK) and NADP-malic enzyme (NADP-ME). Phosphoenol pyruvate carboxykinase and NAD-ME have absolute manganese (Mn) requirements for their activation. As a result, plants that use PEP-CK and NAD-ME decarboxylation pathways may have high Mn requirements. Experiments were carried out to determine the effect of Mn nutritional levels on CO₂ assimilation rate and biomass production of two NAD-ME C4 species: Amaranthus cruentus (purple amaranthus) and Pennisetum glaucum (Pearl millet). Six Mn treatments of 0, 5, 10, 50, 100 and 150 然 were applied. It should be noted that most hydroponic nutrient solutions contain only 1 or 2 然 Mn. Plants were grown hydroponically in growth chambers for 2 weeks and photosynthesis rates on the most recently fully expanded leaves were determined with a LI-COR 6400 infrared gas analyzer. In both species, photosynthesis and shoot dry weight increased with increasing Mn treatment. Pearl millet CO₂ assimilation rates increased by 50 and 188% at 5 and 50 然 Mn, respectively, compared to the control. In purple amaranthus, CO₂ assimilation rates increased by 44 and 50% for plants grown at 50 and 100 然 Mn compared to the control. Pearl millet and purple amaranthus shoot dry weights increased by 53% and 200%, respectively, at 50 and 100 然 Mn concentration, compared to the control. These NAD-ME sub-type C4 plants, pearl millet and purple amaranthus, responded to higher levels of Mn nutrition than normally used in hydroponic media.