Mouse U14 snRNA (previously designated mouse 4.5S hybRNA) is an evolutionarily conserved eukaryotic low molecular weight RNA capable of intermolecular hybridization with both homologous and heterologous 18S rRNA (1). A single genomic fragment of mouse DNA containing the U14 snRNA gene(s) has been isolated from a Charon 4A lambda phage mouse genomic library and sequenced. Results have surprisingly revealed the presence of three U14 snRNA-homologous regions positioned within introns 5, 6, and 8 of the mouse cognate hsc70 heat shock gene. Comparative analysis with the previously reported rat and human cognate hsc70 genes revealed a similar positioning of U14 snRNA-homologous sequences within introns 5, 6 and 8 of the respective rat and human genes. The U14 sequences contained in all three introns of all three organisms are highly homologous to each other and well conserved with respect to the diverging intron sequences flanking each U14-homologous sequence. Comparison of the mouse U14 snRNA sequence with the U14 DNA sequences contained in the three mouse hsc70 introns indicates that intron 5 is utilized for U14 snRNA synthesis in normally growing mouse ascites cells. Analysis of the determined mouse, rat, and human U14-homologous sequences and the upstream and downstream flanking regions did not reveal the presence of any previously defined RNA polymerase I, II, or III binding sites. This suggests that either higher eukaryotic U14 snRNA is transcribed from a unique transcriptional promoter sequence, or alternatively, is generated by intron processing of the hsc70 pre-mRNA transcript.