We previously isolated detergent-resistant membrane complexes (DRMs) that were not solubilized after extraction of Madin-Darby canine kidney cells with Triton X-100 on ice. The complexes were rich in glycosphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins. In this study, we examined the protein composition of DRMs and further characterized the detergent solubility of these structures. Eight to ten cell-surface proteins, including proteins from both apical and basolateral membranes, were recovered in DRMs. Most DRM proteins, however, were not exposed to the surface of whole cells, and we did not detect the complex of cell-surface proteins described by Sargiacomo et al. in a similar study [Sargiacomo, M., et al. (1993) J. Cell Biol. 122, 789-807]. Almost all proteins in DRMs were solubilized by Triton X-100 at temperatures above 30 degrees C or by octyl glucoside on ice. In contrast, a GPI-anchored protein, placental alkaline phosphatase, was mostly solubilized by Triton X-100 after extraction at 10 degrees C. This protein was insoluble in ice-cold Triton X-100 when first delivered to the plasma membrane and remained so for at least 6 h after synthesis. A fraction of the lipids in DRMs remained insoluble after extraction with Triton X-100 at 37 degrees C. DRM lipids were not solubilized by octyl glucoside, suggesting that this detergent selectively extracts proteins from DRMs.