A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to a-amanitin at 1 ,gg/ml but was completely inhibited at 200 ,.g/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to a-amanitin, in contrast to Ul and U2 RNA transcription, which was inhibited >90% at 1 ,ug/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase m transcripts. These results establish that, in contrast to the polymerase 11-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase m.The U series of small nuclear RNAs (snRNAs) consists at present of eight species, U1-U8 (1-3), of which at least three, U1, U2, and U7, are cofactors for messenger RNA processing reactions (2, 4-7). Several lines of evidence indicate that Ul-U5 snRNAs are transcribed by RNA polymerase II (8-16). However, U6 snRNA has features that distinguish it from other U snRNAs. It does not contain a 2,2,7-trimethylguanosine cap structure at its 5' end (17, 18), and it lacks "domain A," a single-stranded region containing the sequence RA(U)nGR (R = purine nucleoside; n > 3) flanked by hairpin structures, which is present in U1, U2, U4, and U5 snRNAs (19).These facts prompted us to investigate the possibility that the transcription of U6 snRNA differs from that of the other snRNAs. In this paper we report the isolation and sequencing of a human U6 snRNA gene and describe experiments demonstrating that U6 snRNA is transcribed by RNA polymerase III. (Brief accounts ofthis work were presented at the