Mouse RNA polymerase I (or A) was purified from an ascites cell line MH134 to virtual homogeneity using a novel purification procedure and examined for subunit composition. In marked contrast to older purifications that reported 5-8 subunits, polymerase I was found to have 11 subunits with remarkable correspondence to those of yeasts. The cDNA encoding a 40-kDa subunit of this enzyme, designated RPA40, was isolated. It predicts a polypeptide of 355 amino acids (M(r) = 40,065) and is encoded by a single copy gene. Protein sequence analysis reveals that RPA40 is the homolog of yeast RPC40, having homology to alpha subunit of Escherichia coli RNA polymerase, yeast RPB3, and human RPB33 RNA polymerase II subunits. The high conservation of this subunit among distant eukaryotes and different RNA polymerases suggests functional importance of this protein as a core subunit.