When Oat1 was first identified as NKT (14), it was argued that it, together with Oct1, represent members of a larger subfamily involved in organic ion and drug transport now known as SLC22. The least studied among SLC22 transporters are the so called unknown substrate transporters (USTs). Here, five novel genes located in a cluster on mouse chromosome 19, situated immediately between Slc22a8 (Oat3)/Slc22a6 (Oat1 or NKT) and Slc22a19 (mouse Oat5), were identified as homologs of the human USTs of the SLC22 family. These genes display preferential expression in liver and kidney, while detailed analyses of one gene, AB056422, identified several splicing variants with differential expression as well as significant embryonic expression. Along with Slc22a6/Oat1, Slc22a8/Oat3 and Slc22a19/Oat5, these Usts constitute the largest known cluster (eight members) of mammalian Slc22 genes. Given the well described functions of the Oats at the ends of this cluster, some of these genes may also be involved in organic anion transport. Ust genes share a signature residue in the potential active site of transmembrane domain 7 that has a uniquely conserved, positively charged, amino acid, Arg356; this may be a site for interaction with organic anion substrates. In certain species examined, Oat1 and Oat3 appeared to be highly conserved, whereas the Ust part of this cluster appeared to undergo repeated species-specific amplification suggesting strong environmental selection pressure, and also suggesting an explanation for copy number variation observed at this locus in humans. A second Ust amplification in mouse appears to be quite recent. However, in some species (e.g. Platypus and Opossum), the amplification seems to have been in Oat members of Slc22. This previously unidentified cluster of Usts may, as suggested for other Slc22 subfamily members, be coordinately regulated and under selective pressure in a species-specific manner.