Abstract: The zinc-binding subunits of yeast RNA polymerase A(I) and B(II) have been identified by a zinc-blotting technique. The two largest subunits of each enzyme (A190, A135, B220, and B150), as well as A12.2, A10, B44.5, B12.6, and B10, bind 65Zn(II). Predicted zinc-binding motifs have been noted in the NH2-terminal part of B220 and the COOH-terminal region of B150 subunits. Subdomains encompassing these motifs have been overproduced as MalE-fusion proteins and shown to retain zinc binding activity. Site-directed mutagenesis in the predicted metal-binding domain of B150 demonstrated its role in zinc binding. Mutations of cysteine residues C1163, C1166, C1182, and C1185 affected 65Zn2+ binding in vitro and caused a lethal or thermosensitive phenotype for growth. The ability to bind zinc is not sufficient for function since mutations in vicinal residues not affecting zinc binding were either lethal or thermosensitive. The role of zinc in RNA polymerase structure and function is discussed in the light of the present results.