The expression of ribosomal protein genes is coordinately regulated in bacteria, yeast, and vertebrates, so that equimolar amounts of ribosomal proteins accumulate for assembly into ribosomes. To understand how expression of ribosomal protein genes is regulated in plants, we altered expression of the large subunit ribosomal protein L3 (RPL3) genes in Nicotiana tabacum using post-transcriptional gene silencing (PTGS). L3 is encoded by two genes, RPL3A and RPL3B, with 80.2% amino acid sequence identity in tobacco. Two types of 'hairpin' RNA (hpRNA) vectors carrying the RPL3A or RPL3B sequences in both sense and antisense orientation were generated in order to alter the expression level of both RPL3 genes. Tobacco plants transformed with a vector containing a 5'-terminal fragment of RPL3A gene displayed decreased RPL3A mRNA levels and a marked increase in the abundance of RPL3B mRNA. These results indicated that expression of the RPL3 genes is coordinately regulated in tobacco. The transgenic plants that contained higher levels of RPL3B mRNA exhibited leaf overgrowth and mottling. Epidermal cells of these plants were increased in number and decreased in size. The precursor rRNA (pre-rRNA) and the mature rRNAs accumulated in these plants, suggesting that ribosome biogenesis is upregulated. Tobacco plants transformed with an hpRNA vector harboring the full-length RPL3B cDNA exhibited efficient silencing of both RPL3A and RPL3B genes, reduced L3 levels, and an abnormal phenotype characterized by a delay in development, stunting, and inhibition of lateral root growth. L3 deficiency led to a reduction in cell number and an increase in cell size, suggesting that L3 positively regulates cell division. Decreasing RPL3 gene expression resulted in a decrease in accumulation of the pre-rRNA, establishing a prominent role for L3 in ribosome biogenesis in plants.