Summary We have cloned and sequenced a DNA fragment from the genome of Drosophila melanogaster which is homologous to Novikoff hepatoma (rat) small nucleolar U3 RNA. DNA sequence analysis shows that the regions of homology between the cloned DNA and rat U3 RNA are distributed over the entire length of the molecule and the total homology of linear sequence is 53%. The present finding supports the generalization that U3 RNA has been evolutionally conserved but diverges between mammals and invertebrates. This clone contains the upstream and downstream sequences required for the efficient and correct transcription of small nuclear RNAs. The secondary structure proposed for the U3 RNA sequence deduced from the cloned DNA is similar in general topography to that reported for rat U3 RNA (Bernstein et al., ref. 23).