A procedure is described for the preparation of metabolically active mesophyll protoplasts from maize, and of functional, intact chloroplasts from these protoplasts. Intact protoplasts show no oxygen evolution with 3-phosphoglycerate or with oxalacetate plus pyruvate as substrates, even when these substances are provided at high concentrations. On the other hand, protoplast extracts and chloroplasts display rates of oxygen evolution of 2-3 µmol min-1 (mg Chl)-1 with the same substances. Pyruvate stimulates oxalacetate-dependent oxygen evolution substantially, indicating good coupling between non-cyclic electron flow and phosphorylation. Low PI concentrations stimulate 3-phosphoglycerate-dependent oxygen evolution; high PI concentrations, and pyridoxal phosphate, inhibit this activity, suggesting a common carrier for 3-phosphoglycerate and PI.