Leaves of Peperomia camptotricha contain three distinct upper tissue layers and a one-cell thick lower epidermis. Light and dark CO(2) fixation rates and the activity of ribulose bisphosphate carboxylase/oxygenase and several C(4) enzymes were determined in the three distinct tissue layers. The majority of the C(4) enzyme activity and dark CO(2) fixation was associated with the spongy mesophyll, including the lower epidermis; and the least activity was found in the median palisade mesophyll. In contrast, the majority of the C(3) activity, that is ribulose bisphosphate carboxylase/oxygenase and light CO(2) fixation, was located in the palisade mesophyll. In addition, the diurnal flux in titratable acidity was greatest in the spongy mesophyll and lowest in the palisade mesophyll. The spatial separation of the C(3) and C(4) phases of carbon fixation in P. camptotricha suggests that this Crassulacean acid metabolism plant may have low photorespiratory rates when it exhibits daytime gas exchange (that is, when it is well watered). The results also indicate that this plant may be on an evolutionary path between a true Crassulacean acid metabolism plant and a true C(4) plant.