Higher plant chloroplast genomes code for a conserved set of 30 tRNAs. This set is believed to be sufficient to support translation, although import of cytosolic tRNA has been proposed to provide additional tRNA species to the chloroplast. Previous knock-outs of tRNA genes, or the pronounced reduction of the level of selected tRNAs, has not led to severe phenotypes. We deleted the two tRNA genes trnN-GUU and trnC-GCA independently from the plastid chromosome of tobacco. No homoplastomic tissue of either DeltatrnN or DeltatrnC plants could be isolated. Both mutants exhibit occasional loss of leaf sectors, and mutant plastid chromosomes are rapidly lost upon relief of selective pressure. This suggests that the knock-out of both trn genes is lethal, and that both tRNA species are required for cell survival. Surprisingly, the impact on chloroplast and cell development differs pronouncedly between the two mutants. Heteroplastomic DeltatrnC and DeltatrnN tissue exhibit different aberrations of the internal membrane systems and, more importantly, heteroplastomic DeltatrnN plants are variegated. Accumulation of tRNA-N and plastid-encoded proteins is reduced in white sectors of DeltatrnN plants, and differentiation of palisade cells is abolished. Our data demonstrate that plastid tRNAs are essential, i.e. not complemented by cytosolic tRNA, and have a differential impact on chloroplast and plant cell development.