When the organic anion transporter Oat1 was first identified as NKT (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, Nigam SK. J Biol Chem 272: 6471-6478, 1997), it was argued that it, together with Oct1, may be part of a larger subfamily (now known as SLC22) involved in organic ion and xenobiotic transport. 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, immediately between Slc22a8 (Oat3)/Slc22a6 (Oat1) and Slc22a19 (Oat5), were identified as homologs of human USTs. These genes display preferential expression in liver and kidney, and one gene, AB056422, has several splicing variants with differential tissue expression and embryonic expression. Along with Slc22a6, Slc22a8, and Slc22a19, these Usts define the largest known cluster of mammalian Slc22 genes. Given the established functions of Oats, these genes may also be involved in organic anion transport. Usts have characteristic motifs and share a signature residue in the possible active site of transmembrane domain 7, a conserved, positively charged, amino acid, Arg356, possibly a site for interaction with organic anions. In certain species, 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 perhaps providing an explanation for copy number variation in the human locus. One Ust amplification in mouse appears to be recent. This cluster may be coordinately regulated and under selective pressure in a species-specific manner.