The full-length mouse RNA polymerase II (pol II) largest subunit (RPB1) gene was used to replace 5070 bp of the yeast Saccharomyces cerevisiae RPB1 gene via homologous recombination and gene replacement in vivo. Transcription of the mouse RPB1 gene using the yeast promoter in the haploid state was confirmed by Northern analysis. This strain of yeast is viable, indicating that mouse RPB1 is able to interact functionally with the other yeast RNA pol II subunits in vivo.