Using cDNA microarrays, we identified StarD4 as a gene whose expression decreased more than 2-fold in the livers of mice fed a high-cholesterol diet. StarD4 expression in cultured 3T3 cells was also sterol-regulated, and known sterol regulatory element binding protein (SREBP)-target genes showed coordinate regulation. The closest homologues to StarD4 were two other StAR-related lipid transfer (START) proteins named StarD5 and StarD6. StarD4, StarD5, and StarD6 are 205- to 233-aa proteins consisting almost entirely of START domains. These three constitute a subfamily among START proteins, sharing approximately 30% amino acid identity with one another, approximately 20% identity with the cholesterol-binding START domains of StAR and MLN64, and less than 15% identity with phosphatidylcholine transfer protein (PCTP) and other START domains. StarD4 and StarD5 were expressed in most tissues, with highest levels in liver and kidney, whereas StarD6 was expressed exclusively in the testis. In contrast to StarD4, expression of StarD5 and MLN64 was not sterol-regulated. StarD4, StarD5, and StarD6 may be involved in the intracellular transport of sterols or other lipids.