Oxysterol-binding proteins (OSBPs) have been described in a wide range of eukaryotes, and are often found to be part of a multi-gene family. We have used bioinformatics and data mining as a starting point for identifying new family members in humans based on the presence of the OSBP signature EQVSHHPP. In addition to OSBP and the recently reported OSBP2, we have found 10 other genes encoding oxysterol-binding domains. Here, we report cDNA and deduced peptide sequences of the previously unknown OSBPs and compare the peptides and genes. All of the genes encode a pleckstrin homology domain, except OSBPL2. However, two of the peptides, OSBPL2 and OSBPL1A, consist of the OSBP domain only. A second OSBPL1 transcript (OSBPL1B) contains 15 additional upstream exons, with a deduced peptide containing a pleckstrin homology domain. Cladistic analysis divides the human OSBP genes into five groups, whose members share similarities in sequence and gene structure; RT-PCR analysis indicates that expression patterns among group members vary widely.