Whole-plant diurnal C exchange analysis provided a noninvasive estimation of daily net C gain in transgenic tobacco (Nicotiana tabacum L.) plants deficient in leaf cytosolic pyruvate kinase (PKc−). PKc− plants cultivated under a low light intensity (100 μmol m−2 s−1) were previously shown to exhibit markedly reduced root growth, as well as delayed shoot and flower development when compared with plants having wild-type levels of PKc (PKc+). PKc− and PKc+ source leaves showed a similar net C gain, photosynthesis over a range of light intensities, and a capacity to export newly fixed 14CO2 during photosynthesis. However, during growth under low light the nighttime, export of previously fixed 14CO2 by fully expanded PKc− leaves was 40% lower, whereas concurrent respiratory 14CO2 evolution was 40% higher than that of PKc+ leaves. This provides a rationale for the reduced root growth of the PKc− plants grown at low irradiance. Leaf photosynthetic and export characteristics in PKc− and PKc+ plants raised in a greenhouse during winter months resembled those of plants grown in chambers at low irradiance. The data suggest that PKc in source leaves has a critical role in regulating nighttime respiration particularly when the available pool of photoassimilates for export and leaf respiratory processes are low.