The gene encoding the mouse Aquaporin-8 water channel protein (<i>Aqp8</i>) was cloned and its genomic structure was defined. <i>Aqp8</i> consists of six exons with boundaries at amino acids 1–4, 5–87, 88–129, 130–201, 202–246 and 247–261 which partially correspond to those of other known aquaporin genes. All splice sites conform to the GT–AG rule except the first one which is GT–GG. Primer extension and RNase protection analyses using mouse liver RNA demonstrated three initiation transcription sites located 385, 156 and 146 bp upstream from the translational start codon. No defined TATA box was found in the 5′-flanking region where numerous CAAT motifs and one GATA box were identified. Fluorescence in situ hybridization localized the <i>Aqp8</i> locus to mouse chromosome 7F3. The 7F region is syntenic with human chromosomes 11, 16 and 10. These results (i) reveal marked structural distinction between the <i>Aqp8</i> gene and the other known mammalian aquaporin genes, (ii) may now permit the molecular characterization of <i>Aqp8</i> expression and (iii) represent a fundamental step for the construction of a target vector to generate transgenic <i>Aqp8</i> knockout mice.