Transgenic Haveria bidentis (a C, species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO, assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO, assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H,O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO, partial pressures. Carbon isotope discrimination was measured concurrently with C O , and H,O exchange on leaves of the control plants and T, progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C, species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO, concentration and CO, leakage from the bundle sheath; however, some down-regulation of the C, cycle also occurred.The C, photosynthetic pathway requires the coordinated functioning of mesophyll and bundle-sheath cells within a leaf. Reactions of the C, cycle concentrate CO, in the bundle-sheath cells for assimilation by Rubisco (Hatch, 1987). This enhances Rubisco carboxylation while at the same time inhibiting Rubisco oxygenation, thus reducing the amount of photorespiration. The energy cost of this C0,-concentrating mechanism is 2 mo1 of ATP per regeneration of 1 mo1 of the primary CO, acceptor PEP, and any CO, that leaks from the bundle sheath rather than being fixed by Rubisco reduces the efficiency of the concentrating mechanism (Hatch, 1987). The compartmentation of the photosynthetic process between mesophyll and bundlesheath cells has complicated the study of C, photosynthesis, and the biophysical characterization of the C0,-concentrating function of C, photosynthesis has been experimentally difficult. Many of the parameters, such as bundle-sheath CO, concentration or the leakiness (4) of the bundle sheath (defined as the fraction of CO, generated by