MAL, BENE and MAL2 are raft-associated integral membrane proteins of the MAL family of proteins involved in membrane trafficking processes. We show here that the human prostate carcinoma PC-3 cell line expresses the transcripts for the three proteins simultaneously. MAL, BENE and MAL2 co-fractionated with caveolin-1 in the raft fraction of PC-3 cells, and immunofluorescence analysis showed colocalization of these proteins with caveolin-1 in a multivesicular intracellular compartment. Markers of the Golgi apparatus, early and recycling endosomes and lipid droplets were excluded from this compartment. Prostate epithelial cells contain vesicular organelles enriched in raft components named prostasomes that are secreted in the prostate fluid. Interestingly, the prostasome fraction isolated from the culture supernatant of PC-3 cells consisted mainly of 30-130 nm cup-shaped vesicles that were positive for MAL, caveolin-1 and CD59, a glycosylphosphatidylinositol-anchored protein previously found in prostasomes. CD63, an integral membrane protein found in multivesicular bodies/lysosomes and secretory granules was also found in PC-3 cell-derived prostasomes. Prostasome secretion was not inhibited by brefeldin A, a compound that blocks the conventional secretory pathway. However, wortmannin, an inhibitor of phosphatidylinositol-3 kinase, reduced the secretion of prostasomes in PC-3 cells. Our results suggest that MAL family proteins are associated with caveolin-1 in a multivesicular compartment that may be involved in prostasomal secretion in PC-3 cells.