The in vitro activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) were measured in cell-free extracts of Platycerium coronarium callus cultured for up to 42 days under photoautotrophic conditions with CO2 enrichment. With an increase in CO2 in the culture environment to 10% (v/v) at low light, the apparent photoautotrophic fixation of CO2 by Rubisco declined, whereas the non-photoautotrophic CO2 fixation by PEPC activity was enhanced. Hence, photosynthesis appears to play a lesser role in providing carbon skeletons and energy with prolonged culture in a CO2-enriched environment. Instead, the anaplerotic supply of C-skeletons by PEPC may be important under such a situation. Short-term H14CO3-fixation experiments indicated that photoautotrophic callus cultured for 3 weeks with 10% CO2 enrichment assimilated less 14CO2 than the control (0.03% CO2). Analyses of 14C-metabolites indicated that about 50% of the total soluble 14CO2 fixed was in the organic acid fraction and 35% in the amino acid fraction. Despite the changes in the in vitro Rubisco/PEPC activity-ratio, no significant change in the 14C distribution pattern was apparent in response to increasing sucrose or CO2 concentrations. The suppression of Rubisco activity and total chlorophyll content in high sucrose or elevated CO2 concentrations suggests an inhibition of the capacity for photoautotrophic callus growth under these conditions.