We have previously isolated a mouse RPA40 (mRPA40) cDNA encoding the 40-kDa subunit of mouse RNA polymerase I and demonstrated that mRPA40 is a mouse homolog of the yeast subunit AC40, which is a subunit of RNA polymerases I and III, having a limited homology to bacterial RNA polymerase subunit alpha (Song, C. Z., Hanada, K., Yano, K., Maeda, Y., Yamamoto, K., and Muramatsu, M. (1994) J. Biol. Chem. 269, 26976-26981). In an extension of the study we have now cloned mouse RPA16 (mRPA16) cDNA encoding the 16-kDa subunit of mouse RNA polymerase I by a yeast two-hybrid system using mRPA40 as a bait. The deduced amino acid sequence shows 45% identity to the yeast subunit AC19 of RNA polymerases I and III, known to associate with AC40, and a local similarity to bacterial alpha subunit. We have shown that mRPA40 mutants failed to interact with mRPA16 and that neither mRPA16 nor mRPA40 can interact by itself in the yeast two-hybrid system. These results suggest that higher eukaryotic RNA polymerase I conserves two distinct alpha-related subunits that function to associate with each other in an early stage of RNA polymerase I assembly.