Chloroplast-encoded large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) are insoluble when separated from the holoenzyme or expressed in Escherichia coli, limiting directed mutagenesis to prokaryotic enzymes. In the present study, we performed directed mutagenesis and chloroplast transformation with the large subunit gene of Chlamydomonas reinhardtii. Two separate mutations were created that are known to influence the CO2/O2 specificity of prokaryotic enzymes. The asparagine 123 to glycine and serine 379 to alanine substitutions gave rise to photosynthesis-deficient mutants that synthesize normal levels of holoenzyme. The Vmax for carboxylation was reduced more than 95% and the Km(CO2) was increased more than 3-fold for both mutant enzymes. Km (O2) was slightly reduced for the glycine 123 enzyme, but increased more than 5-fold for the alanine 379 enzyme. CO2/O2 specificity factors for both enzymes are decreased by more than 70%. Km values for ribulose 1,5-bisphosphate are not significantly affected, but binding affinities for the transition-state analog 2-carboxy-D-arabinitol 1,5-bisphosphate are reduced. The changes brought about by these substitutions in the eukaryotic large subunit are different from the changes observed in prokaryotic enzymes.