Most ribosomal proteins play essential roles in ribosome synthesis and function. In this study, we have analysed the contribution of yeast ribosomal protein L16 to ribosome biogenesis. We show that in vivo depletion of the essential L16 protein results in a deficit in 60S subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability and rapid turnover of early and intermediate pre-60S particles, as evidenced by the reduced steady-state levels of 27SBS and 7SL/S pre-rRNA, and the low amounts of de novo synthesized 27S pre-rRNA and 25S rRNA. Additionally, depletion of L16 blocks nucleocytoplasmic export of pre-60S particles. Moreover, we show that L16 assembles in the nucleolus and binds to early 90S preribosomal particles. Many evolutionarily conserved ribosomal proteins possess extra eukaryote-specific amino- or carboxy-terminal extensions and/or internal loops. Here, we have also investigated the role of the eukaryote-specific carboxy-terminal extension of L16. Progressive truncation of this extension recapitulates, albeit to a lesser extent, the growth and ribosome biogenesis defects of the L16 depletion. We conclude that L16 assembly is a prerequisite to properly stabilize rRNA structures within early pre-60S particles, thereby favouring efficient 27S pre-rRNA processing within the internal transcribed spacer 1 at sites A3 and B1. Upon depletion of L16, the lack of this stabilization aborts early pre-60S particle assembly and subjects these intermediates to turnover.