The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans were subcloned into plasmid pUC9. After induction, both genes were expressed in Escherichia coli and the subunits were assembled into an active holoenzyme. The enzyme was purified from E. coli to high specific activity and was found to contain equimolar amounts of large and small subunits. The assembly of the hexadecameric ribulose bisphosphate carboxylase/oxygenase in E. coli should provide the basis for studies on the mechanism of assembly and the role of small subunits in catalysis.