Eukaryotic RNA polymerases I and III share two distinct alpha-related subunits that show limited homology to the alpha subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of alpha-related subunits in eukaryotes, we have previously identified the alpha-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42+, which encodes the alpha-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42+ encodes a polypeptide with an apparent molecular mass of 42 kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42+ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct alpha-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.