We report the cloning of a cDNA for the mouse unconventional myosin Myo9b, the orthologue of the rat myr5 and human MYOIXb genes. A full-length spleen cDNA of 7087bp encoding a protein of 1961 amino acids was isolated. By RT-PCR, we show that Myo9b is expressed in a wide range of tissues, including heart, brain, muscle and inner ear. In addition, we have identified two alternatively spliced exons. Equivalent exons have not been previously reported for either the human or rat homologues. These exons are located in the Myo9b specific actin-binding site insert of the head domain and in the tail region. A third splice form utilizing an alternative reading frame within the 3'UTR is also described. Several polymorphisms within the coding region were identified; of interest is an in-frame 33bp imperfect duplication within the tail region that was observed only in the C57Bl/6 strain. Myo9b has been previously mapped to mouse chromosome 8 and is a candidate for the mouse mutations myodystrophy and quinky.