The basis for the lesions in the Sp25 and H7 ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) mutants of tobacco was studied in detail because these plants may be suitable hosts for transformation with the genes for Rubisco enzymes of various origins that have different substrate specificities. We show that the Sp25 mutant lacks active holoenzyme, but contains the large and small subunit polypeptides, Rubisco activase and the chloroplast chaperonin, Cpn 60. The large subunit polypeptides were not distributed uniformly in the stroma in the Sp25 mutant as they were in the wild-type plants, but had an anomalous distribution being present only in aggregated clusters notably in chloroplasts with large starch grains. Furthermore, these clusters were not uniformly distributed throughout the photosynthetic cells but were localized largely in the mesophyll cells surrounding the vascular tissue. In contrast to the Sp25 mutant, the H7 mutant contained the Rubisco holoenzyme, but in this case the enzyme was inactive. It is clear that in both these mutants the Rubisco holoenzyme fails to assemble correctly. In the Sp25 mutant assembly is lost completely while in the H7 mutant the holoenzyme is formed, but the assembly process fails to produce an active enzyme. We suggest that the flaw in assembly in the Sp25 mutant results from a defect in chloroplast encoded proteins.