There is increasing evidence for ribosome heterogeneity in biological systems. In Arabidopsis thaliana, the ribosomal protein S15a is encoded by six separate genes, which fall into two evolutionarily distinct categories (Type I and Type II). Type I S15a is a universally conserved component of cytosolic ribosomes, whereas there is ambiguity as to the specific subcellular location of Type II S15a (cytosolic and/or mitochondrial ribosomes). In this study, we investigated the functional significance of the distinct form of ribosomal protein S15a (Type II) in Arabidopsis by examining: the evolutionary relationship of eukaryotic S15a proteins with respect to organellar homologs, the expression of individual Type II S15a genes during various developmental stages by RT-PCR, and the phenotypes of an insertional mutation into the RPS15aE gene. The Type II S15a proteins are plant specific, and the duplication event that gave rise to the Type II S15a genes appears to have occurred during the evolution of land plants. The genes encoding Type II S15a in Arabidopsis are differentially expressed, and mutant plants in which the gene encoding S15aE is knocked down produce larger leaves, longer roots, and possess larger cells than wild-type plants suggesting that the RPS15aE isoform of Type II S15a may act as a regulator of translational activity. Our results add significantly to the understanding of the protein constitution of plant ribosomes and the functional significance of ribosome heterogeneity.