The mouse cathepsin B gene and its flanking regions were cloned and characterized. The gene contains 10 exons and 9 introns spanning about 20 kb. Although the exon-intron organization of the mouse cathepsin B gene showed some similarity to the rat cathepsin H and L genes, significant differences were found. In particular, the highly conserved sequence that contains the catalytically active cysteine in these genes is split at different sites by an intron. As with other thiol proteinases, there is no obvious correspondence between the coding exons and structural or functional units within preprocathepsin B. These results suggest that the lysosomal thiol proteinase genes are evolutionarily ancient and that intron shifting has occurred subsequent to their divergence from a common ancestral form. The 5'-flanking region and exon 1 sequences in the mouse cathepsin B gene have a high GC content of approximately 72%. The 5'-flanking region also contains several potential Sp1 binding sites, but lacks TATA and CAAT motifs. These characteristics suggest that cathepsin B is a "housekeeping" gene and its transcription may be controlled by multiple transcription factors, including Sp1.