The metabolism of ribosomal proteins (r-proteins) and r-protein mRNAs was examined during mouse myoblast differentiation to identify the levels at which r-protein accumulation is regulated. Pulse-chase analyses of r-proteins in myoblasts and fibers indicate that the synthesis of r-proteins is coordinately reduced 2.0-fold following myoblast differentiation and that newly synthesized r-proteins do not turnover. This decreased synthesis of r-proteins in fibers is due to both a reduction in the steady-state levels of r-protein mRNAs and a decrease in the translational efficiency of r-protein mRNAs. Northern analyses of r-protein mRNA indicate that the steady-state levels of r-protein mRNAs S16, L18, and L32 are decreased 1.5-2.0-fold in fibers as compared to myoblasts. Analyses of the distribution of r-protein mRNAs in polysome gradients indicate that their translational efficiencies are reduced 1.3-1.6-fold in fibers as compared to myoblasts. To determine if the decrease in the steady-state levels of r-protein mRNAs is regulated at the level of transcription, the transcription of these genes was measured in isolated nuclei. These experiments show that the transcription of these r-protein genes is reduced 2-6-fold following myoblast differentiation. Thus, the production of r-proteins is regulated both at the level of transcription and translation during mouse myoblast differentiation.