SummaryThe plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The resulting off-white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including¯ower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally,¯attened membranes, reminiscent of stacked thylakoids. The rpo ± material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild-type. Western analysis demonstrates that plastids contain nuclear-coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the DpH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo ± plastids contain functional ribosomes. The detection of the plastidencoded ribosomal protein Rpl2 is consistent with these results. The ®ndings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.