Abstract <p>A study was conducted of the long-term influence of different RL/FRL ratios in the irradiation spectrum of lettuce plants on the activity of a number of reactions of the Benson-Calvin cycle, as well as enzymes of carbon metabolism—chloroplast and cytoplasmic 3-GAP dehydrogenase complexes. Plants were grown for 30 days at a constant photosynthetic photon flux density (180 μmol/m² s) at three RL/FRL ratios = 0.29, 0.89, and 1.67, respectively, and without FRL. Higher rates of net photosynthesis in the absence of FRL in the irradiation spectrum and with an 1.67 RL/FRL ratio, compared to the 0.89 and 0.29 ratios are associated with high activity of the carboxylation reaction, the higher rates of electron transfer in the ETC of chloroplasts and the utilization of triose phosphates. At the same time, high values of 3-GAP dehydrogenase complex activity were noted. Increasing the proportion of FRL in the irradiation spectrum (RL/FRL = 0.29) led to a decrease in the activity of Calvin cycle reactions and the activity of the 3-GAP dehydrogenase complex. The data obtained may indicate a decrease in phosphoglyceric acid (PGA) content, its subsequent reduction to triose phosphates, and a reduced release of triose phosphates into the cytosol. The decrease in the rate of photosynthesis, with a high proportion of FRL in the irradiation spectrum (RL/FRL = 0.29), occurred as a result of a greater reduction in the rate of light-induced photosynthetic reactions than in the activity of dark processes and carbon metabolism enzymes.</p>