Deletion mutants have been derived from a plasmid-cloned repeating unit of Xenopus laevis oocyte 5S DNA by introducing the transposable chloramphenicol-resistance element Tn9 into the AT-rich spacer sequence near the 5' terminus of the X. laevis 5S rRNA gene in a recombinant plasmid and then selecting plasmids which had lost the transposable element. Plasmids lacking the entire transposable element and various portions of the AT-rich spacer sequence flanking the original site of Tn9 integration have been obtained, and their ability to support transcription of the remaining X. laevis 5S rRNA gene has been tested in X. laevis oocyte nuclei. The deletion mutants analyzed in the present study retain the 49 nucleotide nonrepetitive sequence immediately adjacent to the 5' terminus of the gene, but lack as much as 80% of the repetitive AT-rich spacer sequence (Fedoroff and Brown, 1978). Such deletion mutants are fully active templates for 5S rRNA synthesis. This implies that the AT-rich spacer, which comprises half or more of each repeating unit in X. laevis oocyte 5S DNA, is relatively unimportant for correct initiation of transcription, and that if there are extragenic sequences with promoter function, they are likely to reside in the short nonrepetitive region immediately adjacent to the gene.