The gene (POLR2L) encoding a 7.6-kDa subunit (hRPB7.6) of human RNA polymerase has been cloned. It compromises two exons, 116 and 227 bp, respectively, interspaced with an intron of about 2.1 kb. This gene, whose localization has been assigned to the short arm of chromosome 11 (position 11p15), is transcribed in HeLa cells as one major messenger RNA, which encodes a 67-residue polypeptide (7645 Da) that shares strong homologies with the corresponding subunits of other eukaryotic and archaeal RNA polymerase subunits. Like its yeast counterpart (ABC10 beta, encoded by the RPB10 gene), the hRPB7.6 subunit may be shared by all three classes of human nuclear RNA polymerase. Cysteine residues characteristic of an atypical zinc-binding domain are conserved in the homologous sequences of all six species analyzed. A small, related RNA polymerase subunit from vaccinia virus exhibits an identical set of cysteines, suggesting that these residues may be contribute to a crucial function in the multimeric RNA polymerases.