Yeast RNA polymerase I contains 14 distinct polypeptides, including A43, a component of about 43 kDa. The corresponding gene, RPA43, encodes a 326-amino acid polypeptide matching the peptidic sequence of two tryptic fragments isolated from A43. Gene inactivation leads to a lethal phenotype that is rescued by a plasmid containing the 35S ribosomal RNA gene fused to the GAL7 promoter, which allows the synthesis of 35S rRNA by RNA polymerase II in the presence of galactose. A screening for mutants rescued by the presence of GAL7-35SrDNA identified a nonsense rpa43 allele truncating the protein at amino acid position 217. [3H]Uridine pulse labeling showed that this mutation abolishes 35S rRNA synthesis without significant effects on the synthesis of 5 S RNA and tRNAs. These properties establish that A43 is an essential component of RNA polymerase I. This highly hydrophilic phosphoprotein has a strongly acidic carboxyl-terminal domain, and shows no homology to entries in current sequence data banks, including all the genetically identified components of the other two yeast RNA polymerases. RPA43 mapped next to RPA190, encoding the largest subunit of polymerase I. These genes are divergently transcribed and may thus share upstream regulatory elements ensuring their co-regulation.