A genomic clone of the most abundant U1 RNA genes from Xenopus laevis was isolated from erythrocyte DNA and sequenced. Two different U1 RNA genes, U1A and U1B, are encoded in an HindIII 1.5-kb fragment and both are expressed after microinjection in Xenopus oocytes. Deletions and site-directed mutagenesis of the clones revealed two promoter elements in the U1B gene; one, located 250-220 nucleotides upstream from the 5' terminus of mature U1 RNA, functions as an activator, yielding a 10-fold promotion of transcription; the other, located 60-50 nucleotides upstream of the cap site, functions as an essential element for promotion of transcription. The U1A gene contained only the latter element in the cloned fragment. Homologous sequences can be identified in several U RNA genes of X. laevis.