Scavenger receptor class B type I (SR-BI) is a major receptor of the high-density lipoprotein that mediates cholesterol efflux and reverse cholesterol transport. Alternative splicing of the scavenger receptor class B (SR-B) gene is observed and different splice forms, SR-BI and scavenger receptor class B type II (SR-BII), have been shown to function and localize in distinct ways. We have previously shown that SR-B alternative splicing regulation is associated with splicing factor ASF/SF2. In this study, using a SR-B minigene as a model, we determined the critical regulatory regions in the upstream intron, intron 11, by serial deletion and mutation analyses. We also further characterized the regulatory elements in intron 11 as well as in the skipped exon, exon 12. Moreover, we studied the interactions of these elements with the splicing factor ASF/SF2. This study provides new insights into the mechanism of SR-B splicing and it is important for further study on the mechanism of SR-B alternative splicing regulation, such as its regulation by estrogen. (C) 2008 Elsevier Inc. All rights reserved.