Synthesis of yeast 5 S rRNA in a homologous cell-free system has previously been shown to be inhibited by exogenous yeast 5 S rRNA. This inhibition is diminished when 5 S rDNA is first incubated with the cell-free system, implying the presence of a transcription factor IIIA analog in yeast, and is dependent on the nucleotide sequence of the 5 S rRNA added. A two-dimensional gel electrophoresis method was used to demonstrate assembly of newly synthesized 5 S rRNA into a ribonucleoprotein complex containing ribosomal protein YL3. Immunodepletion of the cell-free system with anti-YL3 IgG was found to increase the sensitivity of 5 S rRNA synthesis to inhibition by 5 S rRNA. We propose a model for the regulation of yeast 5 S rRNA synthesis in which YL3 competes with the presumptive transcription factor IIIA analog for binding of 5 S rRNA.