Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10α, Hs10β, Hs8, and Hs6, respectively), homologous to the ABC10α, ABC10β, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10α, Sc10β, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10α and the corresponding Sp10α of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-Δ::HIS3) defective in Sc10α. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10β, Sc10β, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGX(n)CCR motif presumably forms an atypical zinc-binding domain. Hs10β, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-Δ1::HIS3) lacking Sc10β. Hs8 complemented a yeast mutant (rpb8-Δ1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10β cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit.