We have used heterologous RubsiCO genes, expressed from a vector designed in our laboratory, to complement a RubisCO deletion mutant of Rhodobacter capsulatus to growth. Unlike its wild-type parent, the deletion mutant could not grow anaerobically in minimal malate medium in the light (photoheterotrophy), anaerobically in minimal salts medium under a CO₂/H₂ atmosphere (photoautotrophy), or aerobically in the dark on a minimal salts medium under an atmosphere of CO₂/O₂/H₂ (chemoautotrophy) unless it was provided with a functional RubisCO in trans. RubisCO from the cyanobacterium Synechococcus PCC6301 was chosen as a target for mutagenesis because it was unable to complement the deletion strain to low-CO₂ photoautotrophy or to chemoautotrophy. Using the mutator strain Epicurian coli XL1-Red (Stratagene), plasmid libraries of mutant rbcLS genes were generated. When introduced into the host strain, phenotypic differences between the deletion strain complemented with the wild-type Synechococcus enzyme and transconjugates carrying mutated enzymes were used to identify rbcLS genes with potentially interesting mutations. Ten of these mutants were examined in detail. All had apparent changes in RubisCO activity when compared to the wild-type enzyme, and alterations in holoenzyme assembly and protein levels were the most common mutations identified. One mutant enzyme, PAC1, was able to complement to the positive selection conditions of low-CO₂ photoautotrophy and to chemoautotrophy. Although not yet established, the proximity of the PAC1 mutation to the loop 6 region of the protein may have affected key kinetic parameters. We believe this approach of random mutagenesis and bioselection will lead to a deeper understanding of how various residues confer important properties to RubisCO. In contrast to progress made by more traditional structure-function approaches, the molecular basis underlying key kinetic properties such as affinity for CO₂, fallover, and perhaps even specificity for CO₂ over O₂ (W ), should be revealed by studies of this type.