Genetic variability in the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in several Nicotiana species has been characterized by isoelectric focusing patterns. This heritable variation provides an opportunity to examine the functional role of each of these subunits. In this study, specifically designed RuBPCase enzymes composed of identical large subunits but different small subunits were constructed in vivo by interspecific hybridization between the species N. sylvestris, N. tabacum, N. glauca, N. glutinosa, N. plumbaginifolia, and N. tomentosiformis. Small subunit polypeptides were combined to form a sequence of one, two, three, and four polypeptides with the large subunit of N. sylvestris. Kinetic properties of these hybrid enzymes were compared. No differences in the specific activity of either carboxylation or oxygenation nor in Km values for ribulose 1,5-bisphosphate, C02, or 02 were detected among the RuBPCase enzymes from the various interspecific hybrids. Likewise, the ratio of carboxylation to oxygenation was constant.RuBPCase3 is an important photosynthetic enzyme. It catalyzes the key steps in both photosynthesis and photorespiration; the ratio of these two reactions is believed to govern the plant's productivity (6,12,16