By means of the yeast two-hybrid system using the 40-kDa subunit of mouse RNA polymerase I, mRPA40, as the bait, we isolated a mouse cDNA which encoded a protein with significant homology in amino acid sequence to the 12.5-kDa subunit of Saccharomyces cerevisiae RNA polymerase II, B12.5 (RPB11). Specific antibody raised against the recombinant protein that was derived from the cDNA reacted with a 14-kDa polypeptide in highly purified mammalian RNA polymerase II and did not react with any subunit of RNA polymerase I or III. Moreover, the antibody co-immunoprecipitated the largest subunit of mouse RNA polymerase II. These results provide biochemical evidence that the cDNA isolated, named mRPB14, encodes a specific subunit of RNA polymerase II, and indicate that the subunit organization of the enzyme is conserved between yeast and mouse. A possible role of the alpha-motif [Dequard-Chablat, M., Riva, M., Carles, C. and Sentenac, A., J. Biol. Chem. 266 (1991) 15300-15307] in the protein-protein interaction between mRPA40 and mRPB14 is also discussed.