DRM (detergent-resistant membranes), which are resistant to solublization by non-ionic detergents, have been demonstrated to be involved in many key cell functions such as signal transduction, endocytosis and cholesterol trafficking. Covalent modification of proteins by fatty acylation has been proposed to be an important protein-targeting mechanism for DRM association. However, little is known concerning the effects of LCSFA (long-chain saturated fatty acids) on protein composition of DRM in human cancer cells. In the present study, we found that, in Hs578T human breast cancer cells, the major protein increased in DRM in response to the LCSFA stearate (C18:0) was annexin II. Our results demonstrated that annexin II accumulated in DRM specifically in response to physiological concentrations of stearate and palmitate (C16:0), but not long-chain unsaturated fatty acids, in a time- and concentration-dependent manner. This process was reversible and dependent on cholesterol and intracellular calcium. Although calcium was necessary for this translocation, it was not sufficient to induce the annexin II translocation to DRM. We also demonstrate that stearate induced the acylation of caveolin but not that of annexin II. Association of annexin II with caveolin, although not necessarily direct, specifically occurs in DRM in response to stearate. Finally, bromostearate, a stearate analogue that effectively blocks protein acylation, does not induce annexin II translocation to DRM. We conclude that exogenously added LCSFA strongly induces the translocation of annexin II to DRM in Hs578T human breast cancer cells at least partially by association with acylated caveolin.