Proteolipids and complexed acidic phospholipids that cause in vitro hydroxyapatite formation, similarly cause hydroxyapatite deposition in 10-mu pore Millipore chambers when implanted in rabbit muscle pouches. The amount of mineral deposited during a 3-week period, based on the calcium and phosphate contents of the chambers, was directly related to the dry weight of the lipid implanted in the chamber. Chambers containing total lipid extract from rabbit bone from which the complexed acidic phospholipids had been removed, acidic phospholipids from which the the proteolipids had been removed, and empty chambers did not accumulate any detectable mineral during the course of the study. Chambers implanted with synthetic hydroxyapatite served as controls for chemical analyses. The presence of hydroxyapatite in the chambers was established 3 weeks after implantation based on electron microscopic, compositional, and wide-angle X-ray diffraction analyses of the deposits. In the cell-free chambers, lipid-induced hydroxyapatite deposition, but not bone matrix formation occurred. This study demonstrates that proteolipids and complexed acidic phospholipids can cause hydroxyapatite mineral deposition in a physiologic environment. To date, these lipids are the only materials isolated from mineralizing tissues, other than reconstituted collagen, that have been shown capable of causing in vivo mineralization in the absence of cells.