Recently, a new class of histone methyltransferases that plays an indirect role in chromatin silencing by targeting a conserved lysine residue in the nucleosome core was described, namely the Dot1 (disruptor of telomeric silencing) family [Feng, Wang, Ng, Erdjument-Bromage, Tempst, Struhl and Zhang (2002) Curr. Biol. 12, 1052–1058; van Leeuwen, Gafken and Gottschling (2002) Cell (Cambridge, Mass.) 109, 745–756; Ng, Feng, Wang, Erdjument-Bromage, Tempst, Zhang and Struhl (2002) Genes Dev. 16, 1518–1527]. In the present study, we report the isolation, genomic organization and in vivo expression of a mouse Dot1 homologue (mDot1). Expressed sequence tag analysis identified five mDot1 mRNAs (mDot1a–mDot1e) derived from alternative splicing. mDot1a and mDot1b encode 1540 and 1114 amino acids respectively, whereas mDot1c–mDot1e are incomplete at the 5´-end. mDot1a is closest to its human counterpart (hDot1L), sharing 84% amino acid identity. mDot1b is truncated at its N- and C-termini and contains an internal deletion. The five mDot1 isoforms are encoded by 28 exons on chromosome 10qC1, with exons 24 and 28 further divided into two and four sections respectively. Alternative splicing occurs in exons 3, 4, 12, 24, 27 and 28. Northern-blot analysis with probes corresponding to the methyltransferase domain or the mDot1a-coding region detected 7.6 and 9.5 kb transcripts in multiple tissues, but only the 7.6 kb transcript was evident in mIMCD3-collecting duct cells. Transfection of mDot1a–EGFP constructs (where EGFP stands for enhanced green fluorescent protein) into human embryonic kidney (HEK)-293T or mIMCD3 cells increased the methylation of H3-K79 but not H3-K4, -K9 or -K36. Furthermore, DMSO induced mDot1 gene expression and methylation specifically at H3-K79 in mIMCD3 cells in a time- and dose-dependent manner. Collectively, these results add new members to the Dot1 family and show that mDot1 is involved in a DMSO-mediated signal-transduction pathway in collecting duct cells.