Shoot cultures of Hedyotis corymbosa, a C3 species, and sugarcane, a C4 species, were used to examine the effects of various CO2 concentrations and two light intensities on growth and photosynthetic rates. The fresh and dry weights of new growth of Hedyotis shoots were higher when grown under the higher light intensity, while differences among shoots grown under different CO2 levels were marginal. After 14 d of growth in various CO2 concentrations, no significant differences could be observed in the newly produced leaves of Hedyotis with respect to stomatal distribution and number of mesophyll cell layers. Shoots grown under high light intensity did not show higher rates of photosynthesis than those grown under low light intensity. Also, sugarcane shoots grown in a CO2-enriched environment did not have higher photosynthetic rates, perhaps because the C4 pathway is less sensitive to the ambient CO2 concentration. The quantum yield of Hedyotis shoots grown on medium with 20 g l−1 sucrose was lower than that of shoots on lower sucrose concentrations, supporting the view that photosynthesis is inhibited by high levels of sucrose. Our results suggest that Hedyotis shoots in culture exhibit some form of acclimation to high CO2. so that there is no net gain in productivity by photosynthesis.